United States Patent: (10) Patent No.: US 6,171,586 B1

USOO6171586B1
(12) United States Patent (10) Patent No.: US 6,171,586 B1

Lam et al. (45) Date of Patent: Jan. 9, 2001
(54) ANTIBODY FORMULATION WO 94/26302 11/1994 (WO).

WO 96/41164 12/1996 (WO).
(75) Inventors: Xanthe M. Lam, San Francisco; James WO 97/O4807 2/1997 (WO).
WO97/O4801 * 2/1997 (WO).
O. Oeswein, Moss Beach, both of CA WO97/04807 * 2/1997 (WO).
(US); Boonsri Ongpipattanakul, WO 97/04801 2/1997 (WO).
Bangkok (TH); Zahra Shahrokh, San WO97/17087 * 5/1997 (WO).
Francisco; Sharon X. Wang, San WO 97/17087 5/1997 (WO).
Mateo, both of CA (US); Robert P. OTHER PUBLICATIONS
Weissburg, Greenville, DE (US); Rita
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#BIOTEC 2012; Ninth Annual Meeting of the American
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(52) U.S. Cl. ..................................... 424/130.1; 424/152.1; of Surfactant Effects by a Novel EPR Spectroscopic Tech
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Beauvais et al., “Both Glassy State and Native Structure are
(58) Field of Search .............................. 424/130.1, 152.1, Required for Storage Stability of Lyophilized Interleukin-1
424/136.1, 141.1, 1541, 173.1, 133.1; 530/388.75 Receptor Antagonist” Pharm. Res. (Abstract #2007)
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5,736,137 * 4/1998 Anderson et al..
5,770,700 * 6/1998 Webb et al.. Primary Examiner Patrick Nolan
ASSistant Examiner Marianne DiBrino
FOREIGN PATENT DOCUMENTS
(74) Attorney, Agent, or Firm-Lee K. Tan; Wendy M. Lee
30771/89
9/1989 (AU). (57) ABSTRACT
2138853
6/1995 (CA).
303746
2/1989 (EP). A stable aqueous pharmaceutical formulation comprising a
391444
10/1990 (EP).
661060
7/1995 (EP). therapeutically effective amount of an antibody not Sub
WO 89/094O2 10/1989 (WO). jected to prior lyophilization, a buffer maintaining the pH in
WO 89/11297 11/1989 (WO). the range from about 4.5 to about 6.0, a Surfactant and a
WO 90/11091 10/1990 (WO). polyol is described, along with uses for Such a formulation.
WO 92/22653 12/1992 (WO).
WO 94/11026 5/1994 (WO).
WO94/26302 * 11/1994 (WO). 29 Claims, 25 Drawing Sheets
US 6,171,586 B1
Page 2
OTHER PUBLICATIONS Mileski et al., “Inhibition of CD18-dependent neutrophil

adherence reduces organ injury after hemorrhagic shock in
Cleland and Jones, “Development of Stable Protein Formu primates' Surgery 108:206–212 (1990).
lations for Microencapsulation in Biodegradable Polymers'
Proceed. Intern. Symp. Control. Rel. Bioact. Mater. Nielsen, et al., “Stability of Freeze Dried Horseradish Per
22:514–515 (1995). oxidase Conjugated Monoclonal Antibodies Used in Diag
Cleland et al., “The Development of Stable Protein Formu nostic Serology” Journal of Immunoassay 16(2):183-197
lations: A Close Look at Protein Aggregation, Deamidation, (1995).
and Oxidation” Critical Reviews in Therapeutic Drug Car Novak et al., “The tolerance and safety of intravenously
rier Systems 10(4):307-377 (1993). administered benzyl alcohol in methilprednisolone Sodium
Draber et al., “Stability of Monoclonal IgM Antibodies Succinate formulations in normal human Subjects' Toxicol
Freeze-Dried in the Presence of Trehalose” Journal of ogy and Applied Pharmacology 23(1):54-61 (Sep. 1972).
Immunological Methods 181(1):37-43 (1995). Pearlman et al., “Analysis of Protein Drugs’ Peptide and
Hernandez et al., “Role of neutrophils in ischemia-reperfu Protein Drug Delivery, Vincent H. L. Lee, Marcel Dekker,
sion-induced microvascular injury” Am. J. Physiol. 253(3 Pt Inc., Chapter 6, pp. 247-301 (1991).
2):H699-H703 (1987). Picken et al., “Nucleotide Sequence of the gene for heat
Hildreth et al., “The human lymphocyte function-associated stable enterotoxin II of Escherichia coli' Infection and
(HLFA) antigen and a related macrophage differentiation Immunity 42(1):269-275 (1983).
antigen (HMac-1): functional effects of subunit-specific Pikal et al., “The Effects of Formulation Variables on the
monoclonal antibodies” J. Immunol. 134:3272–3280 (1985). Stability of Freeze-Dried Human Growth Hormone”
Hildreth et al., “A Human Lymphocyte-associated Antigen Pharm. Res. 8:427–436 (1991).
Involved in Cell-mediated Lympholysis’ European Journal Pikal, M., “Freeze-Drying of Proteins, Part 2: Formulation
of Immunology 13:202-208 (1983). Selection” Biopharm. 3(9):26-30 (1990).
Hynes, "Integrins: Versatility, modulation, and Signaling in Rao and Kroon, “Orthoclone OKT3: Chemical Mechanisms
cell adhesion” Cell 69(1):11-25 (1992). and Functional Effects of Degradation of a Therapeutic
Izutsu et al., “The effects of additives on the stability of Monoclonal Antibody” Stability and Characterization of
freeze-dried B-galactosidase Stored at elevated tempera Protein and Peptide Drugs. Case Histories, John Wang and
ture” Intl. J. Pharmaceutics 71:137-146 (1991). Rodney Pearlman, New York: Plenum Press pp. 135-158,
Jutila et al., “Inflammation-induced endothelial cell adhe chapter 4, (1993).
Sion to lymphocytes, neutrophils, and monocytes' Trans Reilly et al., “Oral delivery of antibodies: future pharmaco
plantation 48(5):727–731 (1989). kinetic trends' Clin. Pharmocokinet. 32(4):313–323 (1997).
Kossiakoff, A.A., “Tertiary Structure Is a Principal Deter Sapan, “Immunoglobulin stability’ Biotechnol. Appl. Bio
minant to Protein Deamidation” Science 240:191-194
(1988). chem. 25:9-12 (1997).
Lam et al., “Microencapsulation of Recombinant Human Scholtissek et al., “A cloning cartridge of 2 to terminator'
ized Monoclonal Antibody for Local Delivery” Proceedings Nucl. Acids Res. 15(7):3185 (1987).
of the 24th International Symposium on Controlled Release Springer, T., “Adhesion receptors of the immune System”
of Bioactive Materials 24:759–760 (Jun. 15–19, 1997). Nature 346:425–434 (1990).
Lam et al., “Microencapsulation of Recombinant Human Stoolman, L., “Adhesion molecules controlling lymphocyte
ized Monoclonal Antibody for Local Delivery” American migration” Cell 56:907–910 (1989).
Association of Pharmaceutical Scientists, Western Regional Vedder et al., “A Monoclonal Antibody to the Adheren
Meeting, South San Francisco, CA (Abstract #F-22 and ce-promoting Leukocyte Glycoprotein, CD18, Reduces
poster presented at the meeting) (Apr. 24-25, 1997). Organ Injury and Improves Survival from Hemorrhagic
Lam et al., “Pitfalls in Development of Multi-Dose Liquid Shock and Resuscitation in Rabbits' J. Clin. Invest.
Formulations for Three Protein Pharmaceuticals' ACS 81:939–944 (1988).
National Meeting, New Orleans (Abstract #137 and slides Wang et al., “Parenteral Formulations of Proteins and Pep
presented) (Mar. 24-28, 1996). tides: Stability and Stabilizers' J. Parenteral Sci. Tech.
Li et al., “Aggregation and Precipitation of Human Relaxin (Technical Report No. 10) 42(2S):S4–S26 (1988).
Induced by Metal-Catalyzed Oxidation” Biochemistry Yates, “Protein Structure Analysis by Mass Specrometry”
34(17):5762-5772 (1995). Methods in Enzymology 271:351–377 (1996).
Manning et al., “Stability of Protein Pharmaceuticals”
Pharm. Res. 6(11):903–918 (1989). * cited by examiner
U.S. Patent Jan. 9, 2001 Sheet 2 of 25 US 6,171,586 B1
-hydrophobicity
4 15
>
wild 0.5
O X
O do
A.
0 S.
S t
g- -0.5
t
-1
Hydrophilic & Flexible

-15
O 50 100 150 200 FI G 2 A

Amino Acid
hydrophobicity
CDR CDR CDR
2. 0.5
op
e X
A. 0 S.
9 t
r ve
>
I -0.5
-1
Hydrophilic & Flexible

-15
50 100 150 2
Amino Acid "FIG. 2B
0.05
0.04
(0s96u8q–l1OV9yuEa)
FIG. 3A
Storage Temperature (C)

FIG. 3B
0.92 F1 F2 F3 F4 F5
0.9
0.88
([8T1u0/-?6e)| G 86
0.84
0.82
0.8
0.1- OZ Oy 0.1-OZ 08 Oy 0.1-OZ 0/- 0,2- 08 Oy
FIG. 4
0/-OZ 9 E-Oy
40°C
30°C
5°C
-2O°C
70°C
O 5 1O 15 2O 25 30 35 40
Retention Time (Minutes)
FIG. 5A
40°C
3OC
5°C
-20°C
70°C

FIG. 5B

FIG. 6A
30 40
Storage Temperature ( °C)
FIG. 6B
10
0.0031 00032 0.0033 0.0034 0.0035 0.0036 0.0037
1/T (K)
FIG. 7
: H.
E. C 15 20 25
FIG. 8
4OC
:
O 5 10 15 20 25
FIG. 9
RÑ?ONUI Z ?ae! Z
-70
Storage Temper ture (C)
FIG. O
5 O 15 2O 25 30
FIG. 11
92
90
88
E?ILAZOE,z Z - =EZA
N
NN
86
84
|DZUIO-EDZI,
Storage Time (Weeks)

2
N
NN
NN
N
aNLZeoz.Z
EZ Z N=ZoZ.
FIG. 12
0.0031 0.0032 0.0034 0.0035 0.0036 00037

0.0033 1/T (K)
FIG. 13
5 y
-3s M2
S923 NA. Mu 400C
5°C
-20°C
-70°C
0 10 20 30 40 SO 60
FIG. 14A
0 10 20 30 40 50 60
FIG. 14B
10
0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 0.0037
1/T (K)
FIG. 15
U.S. Patent Jan. 9, 2001 Sheet 13 0f 25 US 6,171,586 B1
FIG. 16
1.1
1.05
0.95
0.9
0.85
0.8
100
80 40°C
O-70°C
control
6O
4.0
20
3 4.1 4.1 4. 5 4.5 4.5 5 5 6
100
4056789 :
pH
FIG. 18B
47o C
105
100
95
85
80
Time (weeks)
FIG. 19A
O O 20 30 40 50
Time (weeks)
FIG. 19B
O 10 20 30 40 SO
Time (weeks)
FIG. 19C
ECOR
phoA promoter
STll signal
beta-lactarinase Light Chain
STI signal
Heavy Chain-GCN4 leu zipper
lambda to terminator
Col. 1 origin
tet
FIG. 20
l GAACAACT TCTCCATACT TTGGATAAGG AAATACAGAC AGAAAAATC TCATTGCTGA

6 GTTGTTATTT AAGCTTTGGA GATTATCGTC ACTGCAATGC TTCGCAATAT GGCGCAAAAT
l21 GACCAACAGC GGTTGATTGA TCAGGTAGAG. GGGGCGCTGT ACGAGGTAAA GCCCGATGCC
l3 AGCATTCCTG ACGACGATAC GGAGCTGCTG CGCGATTACG TAAAGAAGTT ATTGAAGCAT
24 CCTCGTCAGT AAAAAGTTAA TCTTTTCAAC AGCTGTCATA AAGTTGTCAC GGCCGAGACT
3O TATAGTCGCT TTGTTTTTAT TTTTTAATGT ATTTGTAACT AGAATTCGAG CTCGCCGGGG
36 ATCCTCTAGA GGTTGAGGTG ATTT AG AAA AAG AA ATC GCA TTT CTT CTT
- 23 M N A F L L
43 GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAC GCT GAT ATC
s A S M F W F S A T N A Y A O
461 CAG AG ACC CAG CC CCG AGC TCC CTG TCC GCC TCT GTG GGC GAT AGG
3. Q M T O S P S S L S A. S W G D R
509 GTC ACC ATC ACC TGT CGT GCC AGT CAG GAC ATC AAC AA TAT CTG AAC
9 w T T C R A O D N N Y L N
557 TGG TAT CAG AAA CCA GGA GCT CCG AAA CTA CTG ATT TAC TAT
P K. w
35 N Y O K P G A L A.
60S ACC TCC ACC CTC CAC TCT GGA GTC CCT TCT CCC TTC TCT GGT TCT GGT
Sl T S L E. S G P S G S
653 TCT GGG ACG GAT TAC ACT CTG ACC ATC AGC AGT CTG CAA CCG GAG GAC
67 S G D Y T L S O P E.
7 Ol TTC GCA ACT TAT TAC TGT CAG CAA GGT AAT ACT CTG CCG CCG ACG TTC
83 F A Y Y C O N L P P T F
749 GGA CAG GGC ACG AAG GTG GAG AC CGA ACT GTG GCT GCA CCA TCT
99 O T K E A P S
797 GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG TCT GGA ACT GCC
15 F S D E G T
845 TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC GTA
3. C N N F A.
89.3 CAG TGG AAG GTG GA AAC GCC CTC CAA TCG GGT AAC TCC CAG GAG AGT
47 W Lu Q S O
94 GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC AGC CTC AGC AGC ACC
l63 W D S D S T Y S L S
989 CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC GCC TGC
179 L. T S K A D Y E. K K W Y
1037 GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC ACA AAG AGC TTC AAC
95 W O C S S P V T K S F N
O85 AGG GGA GAG TGT AA G CTGATCCTCT ACGCCGGACG CATCGTGGCG
2. R G E C
FIG. 21A
Strain Genotype
W3110 K-12F lambda NrrinD-rrTEl
|
1A2 W31 10 Afhua
|
7C1 W3110 AfhuA AphoA A(argF-lac)
16C9 W3110 Afhua AphoA A(argF-lac) deoC
|
23E3 W3110 A?huA AphoA A(argF-lac) deoC AdegP
33B6 W3110 AthuA AphoA A(argF-lac) deoC AdegPilvG
|
49B2 W3110 AthuA AphoA A(argF-lac) deoCAdegP ilvGAfucP
|
49A5 W3110 AfhuA AphoA A(argF-lac) deoCAdegP ilvGAfucP AmalE
FIG. 22
VAL FROM
WORKING CELL BANK
OR
MASTER CELL BANK
PRIMARY INOCULUM, 14-18 hours

SHAKE FLASK temperature controlled
MEDUM
y
7-17 hours
SECONDARY NOCULUM temperature and pH controlled
SECONDARY MEDUM O transferred at 10-25 OD550
PRODUCTION VESSEL, temperature and pH controlled

PRODUCTION MEDIUM Controlled nutrient feeds
harvested at 60-84 hours
w
HARVEST BY CENTRIFUGATION
y
FREEZNG OF CELLS
FIG. 23
U.S. Patent Jan. 9, 2001 Sheet 23 Of 25 US 6,171,586 B1
2.01 102.3
2 100
1.99 97.7
5 1.98 95.5 s
1.97 93.3 s
SS 1.96 91.2 9
C 1.95 89.1
1.94 87.1
1.93 85.1
O O 10 2O 30 40 50 60
Time (days)
f SS
o O
9. X.
S C
N
D 3.
SS
o)
O
-
-10 O 10 20 30 40 50 60
Time (days)
FIG. 24B
mM Treha
FIG. 25
1OOON: 1
250 T. 1 500 T: 1 1 OOOT: 1
Formulation
FIG. 26
KnOZ o] Zso,
U.S. Patent US 6,171,586 B1
100
96
«±---Z Z -
%JeuOou 94
92
90
Formulation
•ØZ Z I
FIG. 27
105
%OJueou
100 200 300 400 50 O 600 700

Time (days)
FIG. 28
US 6, 171586 B1
1 2
ANTIBODY FORMULATION body showed efficacy in reducing multi-organ damage and
death in a rhesus monkey model of decreased cardiac output
RELATED APPLICATION (created by depletion of 2/3 of blood volume for ~2 hours
This is a non-provisional application claiming priority (Mileskietal., Surgery, 108(2):206–212 (1990)). These stud
under Section 119(e) to provisional application 60/053,087, ies point to the therapeutic potential of anti-CD18 antibodies
filed on Jun. 13, 1997, the contents of which are incorpo for acute treatment of hemorrhagic Shock.
rated herein by reference. Another antigen of interest for targeting with antibodies is
the CD20 antigen, also known as “Bp35”. CD20 is a human
FIELD OF THE INVENTION B cell marker which is expressed during early pre-B cell
This invention is directed to a stable aqueous pharmaceu development and remains until plasma cell differentiation.
tical formulation comprising an antibody. The CD20 molecule may regulate a step in the activation
process which is required for cell cycle initiation and
BACKGROUND OF THE INVENTION differentiation and is usually expressed at very high levels on
neoplastic B cells. Thus, the CD20 surface antigen can be
In the past ten years, advances in biotechnology have 15 targeted for treating B cell lymphomas. U.S. Pat. No.
made it possible to produce a variety of proteins for phar 5,736,137 issued Apr. 7, 1998 describes the chimeric anti
maceutical applications using recombinant DNA techniques. body “C2B8” which binds the CD20 antigen and its use to
Because proteins are larger and more complex than tradi treat B cell lymphoma.
tional organic and inorganic drugs (i.e. possessing multiple There is a need in the art for a stable aqueous pharma
functional groups in addition to complex three-dimensional ceutical formulation comprising an antibody, Such as an
Structures), the formulation of Such proteins poses special anti-CD18 or anti-CD20 antibody, which is suitable for
problems. For a protein to remain biologically active, a therapeutic use.
formulation must preserve intact the conformational integ
rity of at least a core Sequence of the protein's amino acids SUMMARY OF THE INVENTION
while at the same time protecting the protein's multiple 25
Accordingly, the invention provides a stable aqueous
functional groups from degradation. Degradation pathways pharmaceutical formulation comprising a therapeutically
for proteins can involve chemical instability (i.e. any process effective amount of an antibody not Subjected to prior
which involves modification of the protein by bond forma lyophilizabon, a buffer maintaining the pH in the range from
tion or cleavage resulting in a new chemical entity) or about 4.5 to about 6.0, a surfactant and a polyol. Preferably
physical instability (i.e. changes in the higher order structure the formulation is stable at a temperature of about 2-8 C.
of the protein). Chemical instability can result from for at least one year, and/or is stable at a temperature of
deamidation, racemization, hydrolysis, oxidation, beta about 30° C. for at least one month and/or is stable following
elimination or disulfide exchange. Physical instability can freezing and thawing of the formulation.
result from denaturation, aggregation, precipitation or The invention also relates to an article of manufacture
adsorption, for example. The three most common protein 35
degradation pathways are protein aggregation, deamidation comprising a container holding a stable aqueous pharma
and oxidation. Cleland et al Critical Reviews in Therapeutic ceutical formulation comprising a therapeutically effective
Drug Carrier Systems 10(4): 307-377 (1993). amount of an antibody not Subjected to prior lyophilizabon,
Included in the proteins used for pharmaceutical applica a buffer maintaining the pH in the range from about 4.5 to
tions are antibodies. An example of an antibody useful for 40 about 6.0, a Surfactant and a polyol.
therapy is an antibody which binds to the CD18 antigen. In yet a further aspect, the invention relates to a method
CD18 is the common B subunit of three heterodimeric for Stabilizing an antibody in an aqueous pharmaceutical
membrane integrins restricted to leukocytes that mediate formulation by combining a therapeutically effective
trafficking and adhesion to the vascular endothelium, par amount of an antibody not Subjected to prior lyophilization,
ticularly at sites of inflammation (for reviews see Hynes, R. 45 a buffer maintaining the pH in the range from about 4.5 to
O. Cell, 69:11–25 (1992); Stoolman, Cell, 56:907–910 about 6.0, a Surfactant and a polyol.
(1989); Jutila et al. Transplantation 48(5): 727-731 (1989); In a still further aspect, the invention concerns a method
Springer, T. A., Nature 346:425-434 (1990); and Albelda of treating a mammal comprising administering a therapeu
and Buck, FASEB.J. 4:2868–2880 (1990)). The heterodimer tically effective amount of the aqueous pharmaceutical for
containing CD18 and CD11b (also called MAC-1) is found 50 mulation disclosed herein to a mammal, wherein the mam
primarily on neutrophils, monocytes, and Some lymphocytes mal has a disorder requiring treatment with the antibody in
whose normal interaction with ICAM-1 on vascular endot the formulation. Where the antibody binds CD18, examples
helium mediates adhesion and “rolling of cells along the of disorders to be treated include hemorrhagic Shock, ther
vasculature. In Severe hemorrhagic trauma with concurrent mal injury (Such as that resulting from burns), Stroke
decrease in cardiac output and ischemia, early (within 30 55 (including ischemic and hemorrhagic stroke) and myocar
min) neutrophil activation (in response to released dial infarction. For an anti-IL8 antibody, disorders to be
cytokines) and up-regulation of MAC-1 increases neutrophil treated include inflammatory disorderS Such as adult respi
"Stickiness'. This precedes extravasation and release of ratory distress syndrome (ARDS), hypovolemic shock,
proteases and Superoxides that ultimately lead to further ulcerative colitis, and rheumatoid arthritis. Where the anti
tissue damage and increased vascular permeability 60 body binds CD20, disorders to be treated include B cell
(Hernandez et al, Am. J. Physiol., 253(3 Pt 2): H699-H703 lymphomas.
(1987)). Reperfusion following resuscitation exacerbates the These and further aspects of the invention will be appar
edema and necrosis, and leads to multi-organ failure and ent to those skilled in the art.
death. Early treatment with monoclonal antibodies to CD18 BRIEF DESCRIPTION OF THE DRAWINGS
in a partally-Severed, ischemic rabbit ear trauma model 65
alleviated tissue necrosis following reattachment (Vedder et FIGS. 1A and 1B depict the amino acid sequence of
al., J. Clin. Invest. 81:939-944 (1988)). A humanized anti rhuMAb CD18 heavy chain (FIG. 1A; SEQ ID NO: 1) and
US 6, 171586 B1
3 4
light chain (FIG. 1B, SEQ ID NO:2). The sequence in italics kcal/mole) was only approximated from these data due to
in FIG. 1A (SEQ ID NO:3) is that of the leucine zipper. the very small K for 5 C., it appears similar to that obtained
FIGS. 2A and 2B are hydroflex plots of rhuMAb CD18 with the SEC and ion-exchange HPLC (IEX) assays.
heavy chain (FIG. 2A) and light chain (FIG. 2B). Kyte FIGS. 14A and 14B show the effect of storage tempera
Dooliutle hydrophobicity calculations averaged with a win ture on the stability of rhuMAb CD18, assayed by IEX, on
dow of 6 amino acids were made on the protein Sequence. formulation F2 at pH 5 (FIG. 14A) and F5 at pH 6 (FIG.
Flexibility values were estimated from the product of the 14B). Two pre-main peaks increased with increasing time
hydrophobicity of each residue and its Side chain Volume and temperature, more pronounced at pH 6. A hump from 22
(again averaged over a window of 6 residues). ASn-Gly and to 28 minutes is an artifact due to an impurity washing off
ASn-Ser motifs in flexible regions are more likely to dea the column.
midate than those in more rigid structures. The CDRs are FIG. 15 is an Arrhenius plot of the % main peak area from
also shown. Most of the heavy chain methionines and heavy IEX of rhuMAb CDI8 formulated in acetate with trehalose
chain ASn 84 are near the CDR's.
FIGS. 3A and 3B show the effect of storage temperature at pH 5 (F2). Activation Energy=20+10 kcal/mole.
FIG.16 shows the three dimensional structure of rhuMAb
on light scattering in different rhuMAb CD18 formulations 15
CD18, including positions of methionine residues. Met 65
after 5 weekS Storage at the designated temperatures as
measured by average absorbance in the range of 340-360 and Met 83 are exposed, whereas others are buried in the
nm (FIG.3A), or the ratio of A278 over A252 nm (FIG.3B). Structure and are expected to be leSS labile to oxidation.
RhuMAb CD18 in formulation F3 is prone to the formation FIG. 17 shows fluorescence spectroscopy of rhuMAb
of insoluble aggregates. CD18 formulations. The emission spectra of various formu
FIG. 4 shows the effect of storage temperature on protein lations were obtained on an SLM8000 fluorimeter, using 280
concentration of rhuMAb CD18 formulations measured by nm excitation wavelength. 500 till Samples were placed in
absorbance at 278 nm corrected for vehicle absorbance at quartz cuveftes and Spectra were obtained with a 2nm.
320 nm. bandwidth at 22 C. Samples were prepared at the different
FIGS. 5A and 5B depict the effect of storage temperature 25 pHS by dilution of a concentrated Stock to 0.1 mg/mL protein
on the stability of rhuMAb CD18 formulation F2 (FIG. 5A) and analyses were made after 24 hr at 25 C. The antibody
and formulation F5 (FIG. 5B) assayed by size exclusion is conformationally stable above pH 3.
chromatography (SEC). A smaller MW species (see arrows) FIGS. 18A and 18B depict the effect of pH and protein
appeared which was more pronounced at pH 6 (FIG. 5B) concentration on the stability of rhuMAb CD18. Formula
compared to pH 5 (FIG. 5A). tions in the pH range of 3 to 6 and concentration range of 0.5
FIGS. 6A and 6B represent the effect of storage time and to 25 mg/mL were placed on stability at 40 C. and -70° C.
temperature on the stability of rhuMAb CD18 showing total for 2 months. Analyses were made by IEX (FIG. 18A) and
peak area (FIG. 6A), and % main peak (FIG. 6B) for all SEC (FIG. 18B). pH 5 was found to be the preferred pH,
formulations, assayed by SEC. No Significant change in total irrespective of protein concentration.
peak area was noted. F1 and F5 maintained the lowest % 35 FIGS. 19A, 19B and 19C depict the kinetics of degrada
main peak. tion of rhuMAb CD18 by SEC (FIG. 19A), IEX (FIG. 19B)
FIG. 7 is an Arrhenius plot of the % main peak area from and MAC-1 binding (FIG. 19C) at various temperatures.
SEC of rhuMAb CD18 formulated in acetate with trehalose Duplicate samples in 10 mM Na acetate, 8% trehalose,
at pH 5 (F2). Activation Energy=19+6 kcal/mole. 0.01% TWEEN 20TM, pH 5 were prepared in 3 cc glass vials
FIG. 8 shows the effect of storage for 5 weeks at 40 C.
40 and placed on Stability at the indicated temperatures. In FIG.
on different rhuMAb CD18 formulations, assayed by hydro 19C, specific activity was the ratio of MAC-1 binding to
phobic interaction chromatography (HIC). The early eluting total F(ab'), ELISA. Samples are stable at 5° C. and 15° C.
peaks at 17.5 min and the shoulder at the leading edge of the up to 43 weeks and at 30° C. up to 1 month. Analyses were
main peak increased compared to controls at -70° C. F2 made at the time points indicated.
45 FIG. 20 depicts the structure of plasmid pS1130 used to
showed the least increase in both components (see FIG. 9).
FIG. 9 shows the effect of storage for 5 weeks at different produce rhuMAb CD18 of the example below.
temperatures on formulation F2, assayed by HIC. A pre FIGS. 21A and 21B depict the full nucleotide sequence
main peak became apparent compared to -70 C. control. (SEQID NO:9) and encoded amino acid sequences (SEQID
FIG. 10 depicts the effect of storage temperature on the 50
No's: 10 and 11, respectively) of the pS 1130 expression
CaSSette.
stability of rhuMAb CD18 (5 week data) assayed by HIC
showing total peak area recovered. A trend towards loSS in FIG. 22 shows derivation of the 49A5 production cell
area was noted in all formulations with increasing Storage line.
temperatures, except for F5 which started lower to begin FIG. 23 is a schematic of the fermentation process for
with. 55 rhuMAb CD18.
FIG. 11 shows the effect of storage for 5 weeks at different FIGS. 24A and 24B depict the effect of pH on the rate of
temperatures on formulation F2, assayed by reverse phase aggregation (FIG. 24A) and oxidation (FIG. 24B) of 40
hydrophobic liquid chromatography (RP-HPLC). A small mg/mL rhuMAb CD20, 25 mM histidine, 0.02% polysorbate
partially resolved pre-main peak component (See arrow) formulations at pH 5.6.5 or 7.5 stored at 40° C.
increased at higher temperatures, while its slightly earlier 60 FIG. 25 depicts the effect of excess molar ratios of
eluting neighbor remained unchanged. trehalose on the freeze-thaw induced aggregation of
FIG. 12 shows the effect of storage for 5 weeks on the rhuMAb CD20 multidose formulations. Each formulation is
stability of rhuMAb CD18 assayed by RP-HPLC showing composed of 40 mg/mL rhuMAb CD20, 20 mM acetate, 0
percentage of the main peak. F2 and F3 showed the highest to 1000:1 molar ratio of trehalose, 0.9% benzyl alcohol and
% main peak after 5 weeks at 40 C. 65 0.02% polysorbate 20, pH 5.0 The amount of trehalose in
FIG. 13 is an Arrhenius plot based on the RP-HPLC 9% formulations 1-4 was follows: 1=0 moles trehalose: 1 mole
main peak for formulation F2. Activation energy (~20 of rhuMAb CD20 (0 mM trehalose); 2=250 moles trehalose:
US 6, 171586 B1
S 6
1 mole rhuMAb CD20 (67 mM trehalose); 3=500 moles period. Preferably, the formulation is stable at room tem
trehalose: 1 mole rhuMAb CD20 (134 mM trehalose); and perature (~30° C.) or at 40° C. for at least 1 month and/or
4=1000 moles trehalose: 1 mole rhuMAb CD20 (267 mM stable at about 2-8 C. for at least 1 year for at least 2 years.
trehalose). Furthermore, the formulation is preferably stable following
FIG. 26 shows the effect of excess molar ratios of freezing (to, e.g., -70 C.) and thawing of the formulation.
trehalose or sodium chloride on the clarity of rhuMAb CD20 A protein "retains its physical Stability” in a pharmaceu
multdose formulations stored at 40 C. for up to four weeks. tical formulation if it shows no signs of aggregation, pre
The composition of the formulations is 40 mg/mL rhuMAb cipitation and/or denaturation upon visual examination of
CD20.20 mM acetate, 0 to 1000:1 molar ratio of trehalose or color and/or clarity, or as measured by UV light Scattering or
Sodium chloride, 0.9% benzyl alcohol and 0.02% polysor by Size exclusion chromatography.
bate 20 at pH 5.0. The clarity of the 500:1 and 1000:1 molar A protein “retains its chemical Stability' in a pharmaceu
ratio of Sodium chloride to rhuMAb CD20 formulations was tical formulation, if the chemical Stability at a given time is
not measured after four weeks at 40 C. due to the physical Such that the protein is considered to Still retain its biological
appearance of these formulations. The 500:1 ratio formula activity as defined below. Chemical stability can be assessed
tion was very opalescent while the 1000:1 ratio formulabon 15
by detecting and quantifying chemically altered forms of the
had Separated into two phases composed of a thin opaque gel protein. Chemical alteration may involve size modification
layer covered with an opalescent fluid. The O.D. of the (e.g. clipping) which can be evaluated using size exclusion
1000:1 molar ratio of Sodium chloride to rhuMAb CD20 chromatography, SDS-PAGE and/or matrix-assisted laser
formulation was 2.72 after two weeks storage at 40 C. desorption ionization/time-of-flight mass spectrometry
FIG. 27 depicts the effect of excess molar ratios of (MALDI/TOF MS), for example. Other types of chemical
trehalose on the stability of rhuMAb CD20 muffidose for alteration include charge alteration (e.g. occurring as a result
mulations stored at 40° C. as analyzed by SEC HPLC. The of deamidation) which can be evaluated by ion-exchange
composition of the formulations is 40 mg/mL rhuMAb chromatography, for example.
CD20, 20 mM acetate, 0–267 mM trehalose, 0.9% benzyl 25
An antibody "retains its biological activity” in a pharma
alcohol and 0.02% polysorbate 20 at pH 5.0. The amount of ceutical formulation, if the biological activity of the anti
trehalose in formulations 1-4 was as follows: 1=0 moles body at a given time is within about 10% (within the errors
trehalose: 1 mole of rhuMAb CD20 (0 mM trehalose); of the assay) of the biological activity exhibited at the time
2=250 moles trehalose: 1 mole rhuMAb CD20 (67 mM the pharmaceutical formulation was prepared as determined
trehalose); 3=500 moles trehalose: 1 mole rhuMAb CD20 in an antigen binding assay, for example. Other “biological
(134 mM trehalose); and 4=1000 moles trehalose: 1 mole activity” assays for antibodies are elaborated hereinbelow.
rhuMAb CD20 (267 mM trehalose). The percent monomer By “isotonic' is meant that the formulation of interest has
at each bnepoint was normalized to the percent monomer at essentially the same osmotic pressure as human blood.
T=0. Isotonic formulations will generally have an osmotic pres
FIG. 28 shows the stability profile of the prototype liquid 35 sure from about 250 to 350 mC)sm. Isotonicity can be
rhuMAb CD20 multidose formulation stored at 2-8 C. for measured using a vapor pressure or ice-freezing type
up to two years as measured by SEC HPLC. The formulation oSmometer, for example.
was composed of 40 mg/mL rhuMAb CD20, 150 mM A "polyol is a Substance with multiple hydroxyl groups,
trehalose, 0.9% benzyl alcohol and 0.02% polysorbate 20 at and includes Sugars (reducing and nonreducing Sugars),
pH 5.0. The percent monomer at each timepoint was nor 40 Sugar alcohols and Sugar acids. Preferred polyols herein
malized to the percent monomer at T=0. The bioactivity of have a molecular weight which is less than about 600 kD
the formulation stored at 2-8 C. for two years was 99.2% (e.g. in the range from about 120 to about 400 kD). A
relative to the reference control as measured by the CDC “reducing Sugar is one which contains a hemiacetal group
asSay. that can reduce metalions or react covalently with lysine and
DETAILED DESCRIPTION OF THE 45 other amino groups in proteins and a “nonreducing Sugar is
PREFERRED EMBODIMENTS
one which does not have these properties of a reducing
Sugar. Examples of reducing Sugars are fructose, mannose,
I. Definitions maltose, lactose, arabinose, Xylose, ribose, rhamnose, galac
tose and glucose. Nonreducing Sugars include Sucrose,
The term “pharmaceutical formulation” refers to prepa 50 trehalose, Sorbose, meleZitose and raffinose. Mannitol,
rations which are in Such form as to permit the biological Xylitol, erythritol, threitol, Sorbitol and glycerol are
actvity of the active ingredients to be unequivocally examples of Sugar alcohols. AS to Sugar acids, these include
effective, and which contain no additional components L-gluconate and metallic salts thereof. Where it desired that
which are toxic to the subjects to which the formulation the formulation is freeze-thaw Stable, the polyol is prefer
would be administered. “Pharmaceutically acceptable” 55 ably one which does not crystallize at freezing temperatures
excipients (vehicles, additives) are those which can reason (e.g. -20° C) such that it destabilizes the antibody in the
ably be administered to a Subject mammal to provide an formulation. Nonreducing SugarS Such as Sucrose and tre
effective dose of the active ingredient employed. halose are the preferred polyols herein, with trehalose being
A “stable” formulation is one in which the protein therein preferred over Sucrose, because of the Superior Solution
essentially retains its physical Stability and/or chemical 60 stability of trehalose.
Stability and/or biological activity upon Storage. Various AS used herein, "buffer” refers to a buffered solution that
analytical techniques for measuring protein Stability are resists changes in pH by the action of its acid-base conjugate
available in the art and are reviewed in Peptide and Protein components. The buffer of this invention has a pH in the
Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, range from about 4.5 to about 6.0; preferably from about 4.8
Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug 65 to about 5.5; and most preferably has a pH of about 5.0.
Delivery Rev. 10: 29-90 (1993), for example. Stability can Examples of buffers that will control the pH in this range
be measured at a Selected temperature for a Selected time include acetate (e.g. Sodium acetate), Succinate (Such as
US 6, 171586 B1
7 8
Sodium Succinate), gluconate, histidine, citrate and other comprising the population are identical except for possible
organic acid buffers. Where a freeze-thaw stable formulation naturally occurring mutations that may be present in minor
is desired, the buffer is preferably not phosphate. amounts. Monoclonal antibodies are highly Specific, being
In a pharmacological Sense, in the context of the present directed against a single antgenic Site. Furthermore, in
invention, a “therapeutically effective amount of an anti contrast to conventional (polyclonal) antibody preparations
body refers to an amount effective in the prevention or which typically include different antibodies directed against
treatment of a disorder for the treatment of which the different determinants (epitopes), each monoclonal antibody
antibody is effective. A “disorder” is any condition that is directed against a single determinant on the antigen. The
modifier "monoclonal” indicates the character of the anti
would benefit from treatment with the antibody. This body as being obtained from a Substantially homogeneous
includes chronic and acute disorders or diseases including population of antibodies, and is not to be construed as
those pathological conditions which predispose the mammal requiring production of the antibody by any particular
to the disorder in question. method. For example, the monoclonal antibodies to be used
A "preservative' is a compound which can be included in in accordance with the present invention may be made by the
the formulation to essentially reduce bacterial action therein, 15 hybridoma method first described by Kohler et al., Nature
thus facilitating the production of a multi-use formulation, 256:495 (1975), or may be made by recombinant DNA
for example. Examples of potential preservatives include methods (see, e.g., U.S. Pat. No. 4,816,567). The “mono
octadecyldimethylbenzyl ammonium chloride, hexametho clonal antibodies' may also be isolated from phage antibody
nium chloride, benzalkonium chloride (a mixture of alkyl libraries using the techniques described in ClackSon et al.,
benzyldimethylammonium chlorides in which the alkyl Nature 352:624–628 (1991) and Marks et al., J. Mol. Biol.
groups are long-chain compounds), and benzethonium chlo 222:581-597 (1991), for example.
ride. Other types of preservatives include aromatic alcohols The monoclonal antibodies herein Specifically include
Such as phenol, butyl and benzyl alcohol, alkyl parabens “chimeric' antibodies (immunoglobulins) in which a portion
Such as methyl or propyl paraben, catechol, resorcinol, of the heavy and/or light chain is identical with or homolo
cyclohexanol, 3-pentanol, and m-creSol. The most preferred 25 gous to corresponding Sequences in antibodies derived from
preservative herein is benzyl alcohol. a particular species or belonging to a particular antibody
As used herein, the term “inflammatory disorders' refers class or Subclass, while the remainder of the chain(s) is
to pathological States resulting in inflammation, e.g. caused identical with or homologous to corresponding Sequences in
by influx of leukocytes and/or neutrophil chemotaxis. antibodies derived from another species or belonging to
Inflammation may result from infection with pathogenic another antibody class or Subclass, as well as fragments of
organisms and Viruses and noninfectious means Such as Such antibodies, So long as they exhibit the desired biologi
trauma or reperfusion following myocardial infarction or cal activity (U.S. Pat. No. 4,816,567; and Morrison et al.,
Stroke, immune response to foreign antigen and autoimmune Proc. Natl. AcadSci, USA 81:6851–6855 (1984)).
responses. Examples of inflammatory disorders include The term “hyperVariable region” when used herein refers
inflammatory skin diseases Such as psoriasis and dermatitis, 35 to the amino acid residues of an antibody which are respon
responses associated with inflammatory bowel disease (Such Sible for antigen-binding. The hyperVariable region com
as Crohn's disease and ulcerative colitis); ischemic reper prises amino acid residues from a “complementarity deter
fusion; adult respiratory distress Syndrome, meningitis, mining region” or “CDR' (i.e. residues 24-34 (L1), 50-56
encephalitis, uvei.S., autoimmune diseases Such as rheuma (L2) and 89–97 (L3) in the light chain variable domain and
toid arthritis, Sorgen’s Syndrome, vasculitis, diseases 40 31-35 (H1), 50–65 (H2) and 95-102 (H3) in the heavy chain
involving leukocyte diapedesis, central nervous System variable domain; Kabat et al., Sequences of Proteins of
(CNS) inflammatory disorder; multiple organ injury Syn Immunological Interest, 5th Ed. Public Health Service,
drome Secondary to Septcaemia or trauma, alcoholic hepa National Institutes of Health, Bethesda, Md. (1991)) and/or
titis, bacterial pneumonia; antigen-antibody complex medi those residues from a “hyperVariable loop'(i.e. residues
ated diseases, hypovolemic shock, glomerulonephritis, 45 26-32 (L1), 50–52 (L2) and 91-96 (L3) in the light chain
multiple Sclerosis, Type I diabetes melitis, acute and delayed variable domain and 26-32 (H1), 53–55 (H2) and 96–101
hyperSensitivity; graft VS. host disease; transplant rejection; (H3) in the heavy chain variable domain; Chothia Lesk J.
reperfusion injury, endotoxic shock, disease States due to Mol. Biol. 196:901–917 (1987)). “Framework” or “FR”
leukocyte dyscrasia and metastasis, asthma, pulmonary oxy residues are those variable domain residues other than the
gen toxicity; inflammation of the lung, including pleurisy, 50 hyperVariable region residues as herein defined. The CDR
alveolitis, vasculitis, pneumonia, chronic bronchitis, and FR residues of the H52 antibody of the example below
bronchiectasis, and cystic fibrosis, etc. are identified in Eigenbrot et al. Proteins. Structure, Func
The term “antibody” is used in the broadest sense and tion and Genetics 18:49-62 (1994).
Specifically covers monoclonal antibodies (including full “Humanized” forms of non-human (e.g., murine) anti
length monoclonal antibodies), polyclonal antibodies, mul 55 bodies are chimeric antibodies which contain minimal
tispecific antibodies (e.g., bispecific antibodies), and anti Sequence derived from non-human immunoglobulin. For the
body fragments So long as they exhibit the desired biological most part, humanized antibodies are human immunoglobu
activity. lins (recipient antibody) in which residues from a hyper
"Antibody fragments' comprise a portion of a full length variable region of the recipient are replaced by residues from
antibody, generally the antigen binding or variable region 60 a hyperVariable region of a non-human species (donor
thereof. Examples of antibody fragments include Fab, Fab', antibody) Such as mouse, rat, rabbit or nonhuman primate
F(ab'), and Fv fragments, diabodies; linear antibodies; having the desired specificity, affinity, and capacity. In Some
Single-chain antibody molecules, and multispecific antibod instances, FR residues of the human immunoglobulin are
ies formed from antibody fragments. replaced by corresponding non-human residues.
The term “monoclonal antibody” as used herein refers to 65 Furthermore, humanized antibodies may comprise residues
an antibody obtained from a population of Substantially which are not found in the recipient antibody or in the donor
homogeneous antibodies, i.e., the individual antibodies antibody. These modifications are made to further refine
US 6, 171586 B1
10
antibody performance. In general, the humanized antibody in that mammal. However, antibodies directed against non
will comprise Substantially all of at least one, and typically polypeptide antigens (such as tumor-associated glycolipid
two, variable domains, in which all or substantially all of the antigens; see U.S. Pat. No. 5,091,178) are also contem
hyperVariable regions correspond to those of a non-human plated.
immunoglobulin and all or substantially all of the FR Where the antigen is a polypeptide, it may be a trans
regions are those of a human immunoglobulin Sequence. The membrane molecule (e.g. receptor) or ligand Such as a
humanized antibody optionally also will comprise at least a growth factor. Exemplary antigens include molecules Such
portion of an immunoglobulin constant region (Fc), typi as renin; a growth hormone, including human growth hor
cally that of a human immunoglobulin. For further details, mone and bovine growth hormone, growth hormone releas
see Jones et al., Nature 321:522–525 (1986); Riechmann et ing factor, parathyroid hormone, thyroid Stimulating hor
al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. mone, lipoproteins, alpha-1-antitrypsin; insulin A-chain;
Biol. 2:593–596 (1992). insulin B-chain; proinsulin, follicle Stimulating hormone;
“Single-chain Fv' or “sv' antibody fragments comprise calcitonin; luteinizing hormone, glucagon; clotting factors
the V and V, domains of antibody, wherein these domains Such as factor VIIIC, factor IX, tissue factor, and von
are present in a single polypeptide chain. Generally, the FV 15 Willebrands factor; anti-clotting factors such as Protein C;
polypeptide further comprises a polypeptide linker between atrial natriuretic factor; lung Surfactant; a plasminogen
the V and V, domains which enables the sRv to form the activator, Such as urokinase or human urine or tSSue-type
desired Structure for antigenbinding. For a review of SFv See plasminogen activator (t-PA), bombesin; thrombin;
Pluckthun in The Pharmacology of Monoclonal Antibodies, hemopoietic growth factor; tumor necrosis factor-alpha and
vol.113, Rosenburg and Moore eds. Springer-Verlag, New -beta; enkephalinase; RANTES (regulated on activation
York, pp. 269-315 (1994). normally T-cell expressed and Secreted); human macrophage
The term “diabodies” refers to small antibody fragments inflammatory protein (MIP-1-alpha); a serum albumin such
with two antigen-binding Sites, which fragments comprise a as human Serum albumin; Muellerian-inhibiting Substance;
heavy chain variable domain (V) connected to a light chain relaxin A-chain, relaxin B-chain; prorelaxin, mouse
25 gonadotropin-associated peptide; a microbial protein, Such
variable domain (V) in the same polypeptide chain as beta-lactamase; DNase; IgE, a cytotoxic T-lymphocyte
(V-V.). By using a linker that is too short to allow pairing asSociated antigen (CTLA), Such as CTLA-4, inhibin;
between the two domains on the same chain, the domains are activin; vascular endothelial growth factor (VEGF); recep
forced to pair with the complementary domains of another tors for hormones or growth factors, protein A or D, rheu
chain and create two antigen-binding sites. Diabodies are matoid factors, a neurotrophic factor Such as bone-derived
described more fully in, for example, EP 404,097; WO neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such
90:6444-6448 (1993). as NGF-?3; platelet-derived growth factor (PDGF); fibroblast
The expression “linear antibodies” when used throughout growth factor such as aFGF and bFGF; epidermal growth
this application refers to the antibodies described in Zapata 35 factor (EGF); transforming growth factor (TGF) such as
et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these TGF-alpha and TGF-beta, including TGF-31, TGF-32,
antibodies comprise a pair of tandem Fd Segments TGF-B3, TGF-B4, or TGF-35; insulin-like growth factor-I
(V-C1-V-C1) which form a pair of antigen binding and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),
regions. Linear antibodies can be bispecific or monospecific. insulin-like growth factor binding proteins; CD proteins
The antibody which is formulated is preferably essentially 40 such as CD3, CD4, CD8, CD19 and CD20; erythropoietin;
pure and desirably essentially homogeneous (i.e. free from Osteoinductive factors, immunotoxins, a bone morphoge
contaminating proteins etc). “Essentially pure' antibody netic protein (BMP); an interferon such as interferon-alpha,
means a composition comprising at least about 90% by -beta, and -gamma; colony Stimulating factors (CSFs), e.g.,
weight of the antibody, based on total weight of the M-CSF, GM-CSF, and G-CSF, interleukins (ILS), e.g., IL-1
composition, preferably at least about 95% by weight. 45 to IL-10, Superoxide dismutase, T-cell receptors, Surface
"Essentially homogeneous' antibody means a composition membrane proteins, decay accelerating factor; viral antigen
comprising at least about 99% by weight of antibody, based Such as, for example, a portion of the AIDS envelope;
on total weight of the composition. transport proteins, homing receptors, addressins, regulatory
“Treatment” refers to both therapeutic treatment and proteins; integrins such as CD11a, CD11b, CD11c, CD18, an
prophylactic or preventative measures. Those in need of 50 ICAM, VLA-4 and VCAM; a tumor associated antigen such
treatment include those already with the disorder as well as as HER2, HER3 or HER4 receptor; and fragments of any of
those in which the disorder is to be prevented. the above-listed polypeptides.
“Mammal’ for purposes of treatment refers to any animal Preferred molecular targets for antibodies encompassed
classified as a mammal, including humans, domestic and by the present invention include CD proteins such as CD3,
farm animals, and Zoo, Sports, or pet animals, Such as dogs, 55 CD4, CD8, CD19, CD20 and CD34; members of the ErbB
horses, cats, cows, etc. Preferably, the mammal is human. receptor family such as the EGF receptor, HER2, HER3 or
II. Modes for Carrying Out the Invention HER4 receptor; cell adhesion molecules such as LFA-1,
Mac1, p150,95, VLA-4, ICAM-1, VCAM and CV/B3 inte
The invention herein relates to a stable aqueous formu grin including either C. or f3 Subunits thereof (e.g. anti
lation comprising an antibody. The antibody in the formu 60 CD11a, anti-CD18or anti-CD11b antibodies); growth fac
lation is prepared using techniques available in the art for tors such as VEGF; an interleukin such as IL8; IgE, blood
generating antibodies, exemplary methods of which are group antigens, flk2/flt3 receptor, obesity (OB) receptor;
described in more detail in the following Sections. mpl receptor, CTLA-4, protein C etc.
The antibody is directed against an antigen of interest. The preferred antibody herein is one which binds to
Preferably, the antigen is a biologically important 65 human CD18 and preferably blocks (partially or completely)
polypeptide and administration of the antibody to a mammal the ability of a cell (e.g. a neutrophil) expressing the CD18
Suffering from a disorder can result in a therapeutic benefit Subunit at its cell Surface to bind to endothelium. Examples
US 6, 171586 B1
11 12
of anti-CD18 antibodies include MHM23 (Hildreth et al., Specifically bind to the protein used for immunization.
Eur: J. Immunol. 13:202-208 (1983)); M18/2 (IgG, Alternatively, lymphocytes may be immunized in vitro.
Sanches-Madrd et al., J. Exp. Med. 158:586 (1983)); H52 Lymphocytes then are fused with myeloma cells using a
(American Type Culture Collection (ATCC) Deposit HB Suitable fusing agent, Such as polyethylene glycol, to form a
10160); Mas191c and IOT18 (Vermot Desroches et al., hybridoma cell (Goding, Monoclonal Antibodies: Principles
Scand. J. Immunol. 33:277–286 (1991)); and NA-8 (WO and Practice, pp.59-103 (Academic Press, 1986)).
94/12214). The preferred antibody is one which binds to the The hybridoma cells thus prepared are Seeded and grown
CD18 epitope to which either MHM23 or H52 binds. In in a Suitable culture medium that preferably contains one or
certain embodiments, the antibody may bind to a region in more substances that inhibit the growth or survival of the
the extracellular domain of CD18 which associates with unfused, parental myeloma cells. For example, if the paren
CD11b and the antibody may also dissociate C. and B chains tal myeloma cells lack the enzyme hypoxanthine guanine
(e.g. the antibody may dissociate the CD11b and CD18 phosphoribosyl transferase (HGPRT or HPRT), the culture
complex as is the case for the MHM23 antibody). medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium),
Techniques for producing antibodies which can be for which substances prevent the growth of HGPRT-deficient
mulated as disclosed herein will be elaborated below. 15 cells.
A. Antibody Preparation Preferred myeloma cells are those that fuse efficiently,
(i) Antigen Preparation support stable high-level production of antibody by the
Soluble antigens or fragments thereof, optionally conju Selected antibody-producing cells, and are Sensitive to a
gated to other molecules, can be used as immunogens for medium Such as HAT medium. Among these, preferred
generating antibodies. For transmembrane molecules, Such myeloma cell lines are murine myeloma lines, Such as those
as receptors, fragments of these (e.g. the extracellular derived from MOPC-21 and MPC-11 mouse tumors avail
domain of a receptor) can be used as the immunogen. able from the Salk Institute Cell Distribution Center, San
Alternatively, cells expressing the transmembrane molecule Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells avail
can be used as the immunogen. Such cells can be derived able from the American Type Culture Collection, Rockville,
from a natural Source (e.g. cancer cell lines) or may be cells 25 Md. USA. Human myeloma and mouse-human heteromy
which have been transformed by recombinant techniques to eloma cell lines also have been described for the production
express the transmembrane molecule. Other antigens and of human monoclonal antibodies (Kozbor, J. Immunol.,
forms thereof useful for preparing antibodies will be appar 133:3001 (1984); Brodeur et al, Monoclonal Antibody Pro
ent to those in the art. duction Techniques and Applications, pp. 51-63 (Marcel
(ii) Polyclonal Antibodies Dekker, Inc., New York, 1987)).
Polyclonal antibodies are preferably raised in animals by Culture medium in which hybridoma cells are growing is
multiple Subcutaneous (sc) or intraperitoneal (ip) injections assayed for production of monoclonal antibodies directed
of the relevant antigen and an adjuvant. It may be useful to against the antigen. Preferably, the binding specificity of
conjugate the relevant antigen to a protein that is immuno monoclonal antibodies produced by hybridoma cells is
genic in the Species to be immunized, e.g., keyhole limpet 35 determined by immunoprecipitation or by an in Vitro binding
hemocyanin, Serum albumin, bovine thyroglobulin, or Soy assay, Such as radioimmunoassay (RIA) or enzyme-linked
bean trypsin inhibitor using a bifunctional or derivatizing immunoabsorbent assay (ELISA).
agent, for example, maleimidobenzoyl SulfoSuccinimide After hybridoma cells are identified that produce antibod
ester (conjugation through cysteine residues), ies of the desired specificity, affinity, and/or activity, the
N-hydroxy Succinimide (through lysine residues), 40 clones may be Subloned by limiting dilution procedures and
glutaraldehyde, succinic anhydride, SOCI, or R'N=C=NR, grown by standard methods (Goding, MonoclonalAntibod
where R and R are different alkyl groups. ies. Principles and Practice, pp.59-103 (Academic Press,
Animals are immunized against the antigen, immuno 1986)). Suitable culture media for this purpose include, for
genic conjugates, or derivatives by combining, e.g., 100 lug example, D-MEM or RPMI-1640 medium. In addition, the
or 5 lug of the protein or conjugate (for rabbits or mice, 45 hybridoma cells may be grown in Vivo as ascites tumors in
respectively) with 3 volumes of Freund's complete adjuvant an animal.
and injecting the Solution intradermally at multiple Sites. The monoclonal antibodies secreted by the subclones are
One month later the animals are boosted with /S to /10 the Suitably Separated from the culture medium, ascites fluid, or
original amount of peptide or conjugate in Freund's com Serum by conventional immunoglobulin purification proce
plete adjuvant by Subcutaneous injection at multiple sites. 50 dures Such as, for example, protein A-Sepharose, hydroxy
Seven to 14 days later the animals are bled and the serum is lapatite chromatography, gel electrophoresis, dialysis, or
assayed for antibody titer. Animals are boosted until the titer affinity chromatography.
plateaus. Preferably, the animal is boosted with the conju DNA encoding the monoclonal antibodies is readily iso
gate of the same antigen, but conjugated to a different lated and Sequenced using conventional procedures (e.g., by
protein and/or through a different cross-linking reagent. 55 using oligonucleotide probes that are capable of binding
Conjugates also can be made in recombinant cell culture as Specifically to genes encoding the heavy and light chains of
protein fusions. Also, aggregating agents. Such as alum are the monoclonal antibodies). The hybridoma cells serve as a
Suitably used to enhance the immune response. preferred source of such DNA. Once isolated, the DNA may
(iii) Monoclonal Antibodies be placed into expression vectors, which are then transfected
Monoclonal antibodies may be made using the hybridoma 60 into host cells Such as E. coli cells, simian COS cells,
method first described by Kohler et al., Nature, 256:495 Chinese hamster ovary (CHO) cells, or myeloma cells that
(1975), or may be made by recombinant DNA methods (U.S. do not otherwise produce immunoglobulin protein, to obtain
Pat. No. 4,816,567). the Synthesis of monoclonal antibodies in the recombinant
In the hybridoma method, a mouse or other appropriate host cells. Recombinant production of antibodies will be
host animal, Such as a hamster or macaque monkey, is 65 described in more detail below.
immunized as hereinabove described to elicit lymphocytes In a further embodiment, antibodies or antibody frag
that produce or are capable of producing antibodies that will ments can be isolated from antibody phage libraries gener
US 6, 171586 B1
13 14
ated using the techniques described in McCafferty et al., process of analysis of the parental Sequences and various
Nature, 348:552–554 (1990). conceptual humanized products using three-dimensional
Clackson et al., Nature, 352:624–628 (1991) and Marks models of the parental and humanized Sequences. Three
etal., J. Mol. Biol., 222:581-597 (1991) describe the isola dimensional immunoglobulin models are commonly avail
tion of murine and human antibodies, respectively, using able and are familiar to those skilled in the art. Computer
phage libraries. Subsequent publications describe the pro programs are available which illustrate and display probable
duction of high affinity (nM range) human antibodies by three-dimensional conformational Structures of Selected can
chain shuffling (Marks et al., Bio/Technology, 10:779-783 didate immunoglobulin Sequences. Inspection of these dis
(1992)), as well as combinatorial infection and in vivo playS permits analysis of the likely role of the residues in the
recombination as a Strategy for constructing very large functioning of the candidate immunoglobulin Sequence, i.e.,
phage libraries (Waterhouse etal., Nuc. Acids. Res., the analysis of residues that influence the ability of the
21:2265-2266 (1993)). Thus, these techniques are viable candidate immunoglobulin to bind its antigen. In this way,
alternatives to traditional monoclonal antibody hybridoma FR residues can be selected and combined from the recipient
techniques for isolation of monoclonal antibodies. and import Sequences So that the desired antibody
The DNA also may be modified, for example, by substi 15 characteristic, Such as increased affinity for the target
tuting the coding Sequence for human heavy- and light-chain antigencs), is achieved. In general, the CDR residues are
constant domains in place of the homologous murine directly and most Substantially involved in influencing anti
sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. gen binding.
Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently Alternatively, it is now possible to produce transgenic
joining to the immunoglobulin coding Sequence all or part of animals (e.g., mice) that are capable, upon immunization, of
the coding Sequence for a non-immunoglobulin polypeptide. producing a full repertoire of human antibodies in the
Typically Such non-immunoglobulin polypeptides are absence of endogenous immunoglobulin production. For
Substituted for the constant domains of an antibody, or they example, it has been described that the homozygous deletion
are Substituted for the variable domains of one antigen of the antibody heavy-chain joining region (J) gene in
combining site of an antibody to create a chimeric bivalent 25 chimeric and germ-line mutant mice results in complete
antibody comprising one antigen-combining Site having inhibifion of endogenous antibody production. Transfer of
Specificity for an antigen and another antigen-combining site the human germ-line immunoglobulin gene array in Such
having Specificity for a different antigen. germ-line mutant mice will result in the production of
(iv) Humanized and Human Antibodies human antibodies upon antigen challenge. See, e.g., Jakobo
A humanized antibody has one or more amino acid vits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993);
residues introduced into it from a Source which is non Jakobovits et al., Nature, 362:255-258 (1993); Brugger
human. These non-human amino acid residues are often mann et al., Year in Immuno., 7:33 (1993); and Duchosal et
referred to as “import” residues, which are typically taken al. Nature 355:258 (1992). Human antibodies can also be
from an “import' variable domain. Humanization can be derived from phage-display libraries (Hoogenboom et al., J.
essentially performed following the method of Winter and 35 Mol. Biol., 227:381 (1991); Marks et al., J. MoL Biol.,
co-workers (Jones et al., Nature, 321:522–525 (1986); 222:581-597 (1991); Vaughan et al. Nature Biotech 14:309
Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen (1996)).
et al., Science, 239:1534–1536 (1988)), by substituting (v) Antibody Fragments
rodent CDRs or CDR sequences for the corresponding Various techniques have been developed for the produc
Sequences of a human antibody. Accordingly, Such "human 40 tion of antibody fragments. Traditionally, these fragments
ized” antibodies are chimeric antibodies (U.S. Pat. No. were derived via proteolytic digestion of intact antibodies
4,816,567) wherein substantially less than an intact human (see, e.g., Morimoto et al., Journal of Biochemical and
variable domain has been Substituted by the corresponding Biophysical Methods 24:107-117 (1992) and Brennan et al.,
Sequence from a non-human species. In practice, humanized Science, 229:81 (1985)). However, these fragments can now
antibodies are typically human antibodies in which Some 45 be produced directly by recombinant host cells. For
CDR residues and possibly some FR residues are substituted example, the antibody fragments can be isolated from the
by residues from analogous sites in rodent antibodies. antibody phage libraries discussed above. Alternatively,
The choice of human variable domains, both light and Fab'-SH fragments can be directly recovered from E. coli
heavy, to be used in making the humanized antibodies is and chemically coupled to form F(ab')2 fragments (Carter et
very important to reduce antigenicity. According to the 50 al., Bio/Technology 10:163–167 (1992)). In another
so-called “best-fit” method, the sequence of the variable embodiment as described in the example below, the F(ab')
domain of a rodent antibody is Screened against the entire is formed using the leucine Zipper GCN4 to promote assem
library of known human variable-domain Sequences. The bly of the F(ab') molecule. According to another approach,
human Sequence which is closest to that of the rodent is then F(ab') fragments can be isolated directly from recombinant
accepted as the human framework (FR) for the humanized 55 host cell culture. Other techniques for the production of
antibody (Sims et al., J. Immunol., 151:2296 (1993); antibody fragments will be apparent to the skilled practitio
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another ner. In other embodiments, the antibody of choice is a Single
method uses a particular framework derived from the con chain Fv fragment (scFv). See WO 93/16185.
Sensus Sequence of all human antibodies of a particular (vi) Multispecific Antibodies
Subgroup of light or heavy chains. The same framework may 60 Multspecific antibodies have binding specificities for at
be used for several different humanized antibodies (Carter et least two different epitopes, where the epitopes are usually
al., Proc. Natl. AcadSci, USA, 89:4285 (1992); Presta et al., from different antigens. While such molecules normally will
J. Immnol., 151:2623 (1993)). only bind two different epitopes (i.e. bispecific antibodies,
It is further important that antibodies be humanized with BSAbs), antibodies with additional specificities Such as
retention of high affinity for the antigen and other favorable 65 trispecific antibodies are encompassed by this expression
biological properties. To achieve this goal, according to a when used herein. Examples of BSAbs include those with
preferred method, humanized antibodies are prepared by a one arm directed against a tumor cell antigen and the other
US 6, 171586 B1
15 16
arm directed against a cytotoxic trigger molecule Such as embodiments when unequal ratioS of the three polypeptide
anti-FeyRI/anti-CD15, anti-p185':/FcyRIII (CD16), anti chains used in the construction provide the optimum yields.
CD3/anti-malignant B-cell (1D10), anti-CD3/anti It is, however, possible to insert the coding Sequences for
p185', anti-CD3/anti-p97, anti-CD3/anti-renal cell two or all three polypeptide chains in one expression vector
carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti when the expression of at least two polypeptide chains in
colon carcinoma), anti-CD3/anti-melanocyte stimulating equal ratioS results in high yields or when the ratios are of
hormone analog, anti-EGF receptor/anti-CD3, anti-CD3/ no particular Significance.
anti-CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18, anti In a preferred embodiment of this approach, the bispecific
neural cell ahesion molecule (NCAM)/anti-CD3, anti-folate antibodies are composed of a hybrid immunoglobulin heavy
binding protein (FBP)/anti-CD3, anti-pan carcinoma asso chain with a first binding Specificity in one arm, and a hybrid
ciated antigen (AMOC-31)/anti-CD3; BSAbs with one arm immunoglobulin heavy chain-light chain pair (providing a
which binds Specifically to a tumor antigen and one arm Second binding specificity) in the other arm. It was found
which binds to a toxin Such as anti-Saporin/anti-ld-1, anti that this asymmetric structure facilitates the Separation of the
CD22/anti-saporin, anti-CD7/anti-saporin, anti-CD38/anti desired bispecific compound from unwanted immunoglobu
Saporin, anti-CEA/anti-ricin A chain, anti-interferon-C.(IFN 15 lin chain combinations, as the presence of an immunoglo
O)/anti-hybridoma idiotype, anti-CEA/anti-Vinca alkaloid; bulin light chain in only one half of the bispecific molecule
BSAbS for converting enzyme activated prodrugs. Such as provides for a facile way of Separation. This approach is
anti-CD30/anti-alkaline phosphatase (which catalyzes con disclosed in WO94/04690. For further details of generating
version of mitomycin phosphate prodrug to mitomycin bispecific antibodies See, for example, Suresh et al., Methods
alcohol); BSAbs which can be used as fibrinolytic agents in Enzymology, 121:210 (1986).
Such as anti-fibrin/anti-tSSue plasminogen activator (tPA), According to another approach described in WO96/
anti-flbrin/anti-urokinase-type plasminogen activator (uPA); 27011, the interface between a pair of antibody molecules
BSAbS for targeting immune complexes to cell Surface can be engineered to maximize the percentage of het
receptorS Such as anti-low density lipoprotein (LDL)/anti-Fc erodimers which are recovered from recombinant cell cul
receptor (e.g. FcyRI, FcyRII or FcyRIII); BSAbs for use in 25 ture. The preferred interface comprises at least a part of the
therapy of infectious diseases Such as anti-CD3/anti-herpes C3 domain of an antibody constant domain. In this method,
simplex virus (HSV), anti-T-cell receptor: CD3 complex/ one or more Small amino acid Side chains from the interface
anti-influenza, anti-FcyR/anti-HIV, BSAbs for tumor detec of the first antibody molecule are replaced with larger Side
tion in vitro or in vivo Such as anti-CEA/anti-EOTUBE, chains (e.g. tyrosine or tryptophan). Compensatory "cavi
anti-CEA/anti-DPTA, anti-p185'/anti-hapten; BSAbs as ties” of identical or similar size to the large side chain(s) are
vaccine adjuvants, and BSAbS as diagnostic tools Such as created on the interface of the Second antibody molecule by
anti-rabbit IgG/anti-ferritin, anti-horse radish peroxidase replacing large amino acid Side chains with Smaller ones
(HRP)/anti-hormone, anti-Somatostatin/anti-Substance P, (e.g. alanine or threonine). This provides a mechanism for
anti-HRP/anti-FITC, anti-CEA/anti-B-galactosidase. increasing the yield of the heterodimer over other unwanted
Examples of trispecific antibodies include anti-CD3/anti 35 end-products Such as homodimerS.
CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 and anti Bispecific antibodies include cross-linked or "heterocon
CD3/anti-CD8/anti-CD37. Bispecific antibodies can be pre jugate' antibodies. For example, one of the antibodies in the
pared as full length antibodies or antibody fragments (e.g. heteroconjugate can be coupled to avidin, the other to biotin.
F(ab') bispecific antibodies). Such antibodies have, for example, been proposed to target
Methods for making bispecific antibodies are known in 40 immune system cells to unwanted cells (U.S. Pat. No.
the art. Traditional production of full length bispecific anti 4,676.980), and for treatment of HIV infection (WO
bodies is based on the coexpression of two immunoglobulin 91/00360, WO92/200373, and EP 03089). Heteroconjugate
heavy chain-light chain pairs, where the two chains have antibodies may be made using any convenient croSS-linking
different specificities (Millstein et al., Nature, 305:537-539 methods. Suitable cross-linking agents are well known in the
(1983)). Because of the random assortment of immunoglo 45 art, and are disclosed in U.S. Pat. No. 4,676,980, along with
bulin heavy and light chains, these hybridomas (quadromas) a number of croSS-linking techniques.
produce a potential mixture of 10 different antibody Techniques for generating bispecific antibodies from anti
molecules, of which only one has the correct bispecific body fragments have also been described in the literature.
structure. Purification of the correct molecule, which is For example, bispecific antibodies can be prepared using
usually done by affinity chromatography Steps, is rather 50 chemical linkage. Brennan et al., Science, 229: 81 (1985)
cumberSome, and the product yields are low. Similar pro describe a procedure wherein intact antibodies are pro
cedures are disclosed in WO93/08829, and in Traunecker et teolytically cleaved to generate F(ab')2 fragments. These
al., EMBO J., 10:3655-3659 (1991). fragments are reduced in the presence of the dithiol com
According to a different approach, antibody variable plexing agent Sodium arsenite to Stabilize vicinal dithiols
domains with the desired binding specificities (antibody 55 and prevent intermolecular disulfide formation. The Fab'
antigen combining sites) are fused to immunoglobulin con fragments generated are then converted to thionitrobenzoate
Stant domain Sequences. The fusion preferably is with an (TNB) derivatives. One of the Fab'-TNB derivatives is then
immunoglobulin heavy chain constant domain, comprising reconverted to the Fab'-thiol by reduction with mercaptoet
at least part of the hinge, CH2, and CH3 regions. It is hylamine and is mixed with an equimolar amount of the
preferred to have the first heavy-chain constant region 60 other Fab'-TNB derivative to form the bispecific antibody.
(CH1) containing the site necessary for light chain binding, The bispecific antibodies produced can be used as agents for
present in at least one of the fusions. DNAS encoding the the Selective immobilization of enzymes.
immunoglobulin heavy chain fusions and, if desired, the Recent progreSS has facilitated the direct recovery of
immunoglobulin light chain, are inserted into Separate Fab'-SH fragments from E. coli, which can be chemically
expression vectors, and are co-transfected into a Suitable 65 coupled to form bispecific antibodies. Shalaby et al., J. Exp.
host organism. This provides for great flexibility in adjusting Med., 175: 217-225 (1992) describe the production of a
the mutual proportions of the three polypepbde fragments in fully humanized bispecific antibody F(ab') molecule. Each
US 6, 171586 B1
17 18
Fab' fragment was separately Secreted from E. coli and the Fc domain are transferred. Still more preferred, the
Subjected to directed chemical coupling in vitro to form the epitope is taken from the CH2 domain of the Fc region (e.g.,
bispecific antibody. of an IgG) and transferred to the CH1, CH3, or V region,
Various techniques for making and isolating bispecific or more than one Such region, of the antibody. Alternatively,
antibody fragments directly from recombinant cell culture the epitope is taken from the CH2 domain of the Fc region
have also been described. For example, bispecific antibodies and transferred to the C, region or V, region, or both, of the
have been produced using leucine ZipperS. Kostelny et al., J. antibody fragment.
Immunol., 148(5):1547–1553 (1992). The leucine zipper In one most preferred embodiment, the Salvage receptor
pepbdes from the Fos and Jun proteins were linked to the binding epitope comprises the sequence (5' to 3'): PKNS
Fab" portions of two different antibodies by gene fusion. The SMISNTP(SEQID NO:4), and optionally further comprises
antibody homodimers were reduced at the hinge region to a Sequence Selected from the group consisting of HQSLGTO
form monomers and then re-oxidized to form the antibody (SEQ ID NO:5), HQNLSDGK (SEQ ID NO:6), HQNIS
heterodimers. This method can also be utilized for the DGK (SEQ ID NO:7), or VISSHLGQ (SEQ ID NO:8),
production of antibody homodimers. The “diabody” tech
particularly where the antibody fragment is a Fab or F(ab').
In another most preferred embodiment, the Salvage receptor
nology described by Hollinger et al., Proc. Nati. Acad. Sci. 15 binding epitope is a polypeptide containing the Sequence(s)
USA, 90:6444-6448 (1993) has provided an alternative (5' to 3'): HQNLSDGK (SEQ ID NO:6), HQNISDGK (SEQ
mechanism for making bispecific antibody fragments. The ID NO:7), or VISSHLGQ (SEQ ID NO:8) and the sequence:
fragments comprise a heavy-chain variable domain (V) PKNSSMISNTP (SEQ ID NO:4).
connected to a light-chain variable domain (V) by a linker (ix) Other Covalent Modifications of the Antibody
which is too short to allow pairing between the two domains Covalent modifications of the antibody are included
on the same chain. Accordingly, the V and V, domains of within the scope of this invention. They may be made by
one fragment are forced to pair with the complementary V, chemical Synthesis or by enzymatic or chemical cleavage of
and V. domains of another fragment, thereby forming two the antibody, if applicable. Other types of covalent modifi
antigen-binding sites. Another Strategy for making bispecific cations of the antibody are introduced into the molecule by
antibody fragments by the use of single-chain Fv (sEv) 25 reacting targeted amino acid residues of the antibody with an
dimerS has also been reported. See Gruber et al., J. Immunol, organic derivatizing agent that is capable of reacting with
152:5368 (1994). Selected side chains or the N- or C-terminal residues.
Antibodies with more than two Valencies are contem Examples of covalent modifications are described in U.S.
plated. For example, trispecific antibodies can be prepared. Pat. No. 5,534,615, specifically incorporated herein by ref
Tuft et al. J. Immunol. 147: 60 (1991). erence. A preferred type of covalent modification of the
(vii) Effector Function Engineering antibody comprises linking the antibody to one of a variety
It may be desirable to modify the antibody of the inven of nonproteinaceous polymers, e.g., polyethylene glycol,
tion with respect to effector function, So as to enhance the polypropylene glycol, or polyoxyalkylenes, in the manner
effectiveness of the antibody. For example cysteine residue set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
(s) may be introduced in the Fc region, thereby allowing 35 4,670,417; 4,791,192 or 4,179,337.
interchain disulfide bond formation in this region. The (x) Selecting Biologically Active Antibodies
homodimeric antibody thus generated may have improved Antibodies produced as described above may be Subjected
internalization capability and/or increased complement to one or more “biological activity' assays to Select an
mediated cell killing and antibody-dependent cellular cyto antibody with beneficial properties from a therapeutic per
toxicity (ADCC). See Caron et al., J. Exp Med. 40 Spective.
176: 1191–1195 (1992) and Shopes, B. J. Immunol. The antibody may be screened for its ability to bind the
148:2918–2922 (1992). Homodimeric antibodies with antigen against which it was raised. In the case of an
enhanced anti-tumor activity may also be prepared using anti-CD18 antibody, as shown in the example below, the
heterobifunctional cross-linkers as described in Wolff et al. antigen binding properties of the antibody can be evaluated
Cancer Research 53:2560-2565 (1993). Alternatively, an 45 in a “MAC-1 capture assay”. Briefly, the assay involves first
antibody can be engineered which has dual Fc regions and coating ELISA plates with an anti-CD18 antibody that binds
may thereby have enhanced complement lysis and ADCC to a site of MAC-1 different from the anti-CD18 antibody of
capabilities. See Stevenson et al Anti-Cancer Drug Design interest to capture a recombinant preparation of Soluble
3:219–230 (1989). MAC-1, followed by a wash, addition of the sample to be
(viii) Antibody-Salvage Receptor Binding Epitope 50 tested, wash, addition of goat HRP-labeled anti-human
Fusions. F(ab') antibody and colonmetric detection of OPD sub
In certain embodiments of the invention, it may be strate. The total amount of antibody may be measured by
desirable to use an antibody fragment, rather than an intact first coating an ELISA plate with a polyclonal anti-F(ab')
antibody, to increase tumor penetration, for example. In this antibody, followed by addition of the sample, and then
case, it may be desirable to modify the antibody fragment in 55 HRP-anti-F(ab') and colorimetric detection of the HRP
order to increase its serum half life. This may be achieved, substrate, OPD. Specific activity is the ratio of the MAC-1
for example, by incorporation of a Salvage receptor binding binding to total F(ab'), ELISA value.
epitope into the antibody fragment (e.g. by mutation of the In another embodiment, the affinity of the antibody may
appropriate region in the antibody fragment or by incorpo be determined by Saturation binding; ELISA; and/or com
rating the epitope into a peptide tag that is then fused to the 60 petition assays (e.g. RIAS), for example.
antibody fragment at either end or in the middle, e.g., by Also, the antibody may be Subjected to other biological
DNA or peptide synthesis). activity assays, e.g., in order to evaluate its effectiveness as
The Salvage receptor binding epitope preferably consti a therapeutic. Such assays are known in the art and depend
tutes a region wherein any one or more amino acid residues on the target antigen and intended use for the antibody.
from one or two loops of a Fc domain are transferred to an 65 Where the antibody binds CD18, examples of biological
analogous position of the antibody fragment. Even more activity assays include a slide adhesion assay, phagocytosis
preferably, three or more residues from one or two loops of assay, neutrophil binding assay and degranulation assay.
US 6, 171586 B1
19 20
The slide adhesion assay involves preincubating heparin Klebsiella, Proteus, Salmonella, e.g., Salmonella
ized blood with various concentrations of anti-CD18 typhimurium, Serrafia, e.g., Serratia marceScans, and
antibody, and then placing aliquots onto chambered glass Shigeila, as well as Bacilli such as B. Subtilis and B.
slides. The slides are incubated at 37 C. to allow cells to licheniformis (e.g., B. licheniformis 41P disclosed in DD
adhere, nonadherent cells are gently washed off, the adher 266,710 published Apr. 12, 1989), Pseudomonas such as P.
ent cells are Stained, and the average number of adherent aeruginosa, and Streptomyces. One preferred E. coli cloning
cells per microscope field is determined for 30 fields. host is E. coli 294 (ATCC 31,446), although other strains
For the phagocytosis assay, heparinized whole blood is such as E. coli B, E. coli X 1776 (ATCC 31,537), and Ecoil
obtained and the red blood cells are lysed. The cells are then W3110 (ATCC 27,325) are suitable. These examples are
incubated with opsonized BODIPY-labeled Staphylococcus illustrative rather than limiting.
aureus particles for 30 min at 37 C. The cells are then In additlon to prokaryotes, eukaryotic microbes Such as
analyzed by FACS, and the fluorescence intensity in the filamentous fungi or yeast are Suitable cloning or expression
hosts for antibody-encoding vectors. Saccharomyces
neutrophil gate is determined as an indication of the extent cerevisiae, or common baker's yeast, is the most commonly
of phagocytosis. used among lower eukaryotic host microorganisms.
For determining binding of anti-CD18 antibody to 15
However, a number of other genera, Species, and Strains are
neutrophils, whole blood is incubated with various concen commonly available and useful herein, Such as Schizosac
trations of the anti-CD18 antibody. The cells are then stained charomyces pombe, Kluyveromyces hosts Such as, e.g., K.
with a FITC-conjugated goat anti-human F(ab') antibody, lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC
the red blood cells are lysed, and the white blood cells are 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC
analyzed by FACS. The fluorescence intensity in the neu 56,500), K. drosophilarum (ATCC 36,906), K.
trophil gate is proportional to the extent of anti-CD18 thermotolerans, and K. marxianus, yarrowia (EP402.226);
antibody binding. Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
For a degranulation assay, neutrophils are isolated from (EP244,234); Neurospora crassa; Schwanniomyces such as
whole blood and preincubated with anti-CD18 antibody. The Schwanniomyces Occidentalis, and filamentous fungi Such
cells are then Stimulated with opSonized Zymosan particles 25 as, e.g., Neurospora, Penicillium, Tolypocladium, and
and allowed to Stand at room temperature. The cellular Aspergillus hosts Such as A. nidulans and A. niger.
Supernatants are then collected, and degranulation is Suitable host cells for the expression of glycosylated
assessed either by Specific ELISA (for myeloperoxidase or antibody are derived from multicellular organisms.
lactoferrin) or by enzyme assay (for elastase). Examples of invertebrate cells include plant and insect cells.
For other antibodies, examples of biological activity Numerous baculoviral Strains and variants and correspond
assays include tumor cell growth inhibition assays (as ing permissive insect host cells from hosts Such as
described in WO 89/06692, for example); antibody Spodoptera frugiperda (caterpillar), Aedes aegypti
dependent cellular cytotoxicity (ADCC) and complement (mosquito), Aedes albopictus (mosquito), Drosophila mela
mediated cytotoxicity (CDC) assays (U.S. Pat. No. 5,500, nogaster (fruitfly), and Bombyx mori have been identified. A
362); and agonistic activity or hematopoiesis assays (See 35 variety of Viral Strains for transfection are publicly available,
WO95/27062). e.g., the L-1 variant of Autographa californica NPV and the
To Screen for antibodies which bind to a particular epitope Bm-5 strain of Bombyx mori NPV, and such viruses may be
on the antigen of interest (e.g., those which block binding of used as the virus herein according to the present invention,
the humanized H52 antibody of the example to CD18), a particularly for transfection of Spodoptera frugiperda cells.
routine cross-blocking assay Such as that described in 40 Plant cell cultures of cotton, corn, potato, Soybean, petunia,
Antibodies, A Laboratory Manual, Cold Spring Harbor tomato, and tobacco can also be utilized as hosts.
Laboratory, Ed Harlow and David Lane (1988), can be However, interest has been greatest in Vertebrate cells,
performed. Alternatively, epitope mapping, e.g. as described and propagation of vertebrate cells in culture (tissue culture)
in Champe et al., J. Biol. Chem. 270: 1388–1394 (1995), can has become a routine procedure. Examples of useful mam
be performed to determine whether the antibody binds an 45 malian host cell lines are monkey kidney CV1 line trans
epitope of interest. formed bySV40 (COS-7, ATCC CRL 1651); human embry
B. Vectors, Host Cells and Recombinant Methods onic kidney line (293 or 293 cells subloned for growth in
For recombinant production of the antibody, the nucleic suspension culture, Graham et al., J. Gen Virol. 36:59
acid encoding it is isolated and inserted into a replicable (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
vector for further cloning (amplification of the DNA) or for 50 Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al.,
expression. DNA encoding the monoclonal antibody is Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli
readily isolated and Sequenced using conventional proce cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
dures (e.g., by using oligonucleotide probes that are capable monkey kidney cells (CV1 ATCC CCL 70); African green
of binding specifically to genes encoding the heavy and light monkey kidney cells (VERO-76, ATCC CRL-1587); human
chains of the antibody). Many vectors are available. The 55 cervical carcinoma cells (HELA, ATCC CCL 2); canine
vector components generally include, but are not limited to, kidney cells (MDCK, ATCCCCL34); buffalo rat liver cells
one or more of the following: a signal Sequence, an origin of (BRL 3A, ATCC CRL 1442); human lung cells (W138,
replication, one or more marker genes, an enhancer element, ATCC CCL 75); human liver cells (Hep G2, HB 8065);
a promoter, and a transcription termination sequence (e.g. as mouse mammary tumor (MMT 060562, ATCC CCL51);
described in U.S. Pat. No. 5,534,615, specifically incorpo 60 TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68
rated herein by reference). (1982)); MRC5 cells; FS4 cells; and a human hepatoma line
Suitable host cells for cloning or expressing the DNA in (Hep G2).
the vectors herein are the prokaryote, yeast, or higher Host cells are transformed with the above-described
eukaryote cells described above. Suitable prokaryotes for expression or cloning vectors for antibody production and
this purpose include eubacteria, Such as Gram-negative or 65 cultured in conventional nutrient media modified as appro
Gram-positive organisms, for example, Enterobacteriaceae priate for inducing promoters, Selecting transformants, or
Such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, amplifying the genes encoding the desired Sequences.
US 6, 171586 B1
21 22
The host cells used to produce the antibody of this C. Preparation of the Formulation
invention may be cultured in a variety of media. Commer After preparation of the antibody of interest as described
cially available media such as Ham's F10 (Sigma), Minimal above, the pharmaceutical formulation comprising it is
Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), prepared. The antibody to be formulated has not been
and Dulbecco's Modified Eagle's Medium (DMEM), Subjected to prior lyophilization and the formulation of
Sigma) are Suitable for culturing the host cells. In addition, interest herein is an aqueous formulation. Preferably the
any of the media described in Ham et al., Meth. Enz. 58:44 antibody in the formulabon is an antibody fragment, Such as
(1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. an F(ab'), in which case problems that may not occur for the
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or full length antibody (such as clipping of the antibody to Fab)
5,122,469, WO 90/03430; WO 87/00195; or U.S. Pat. No. may need to be addressed. The therapeutically effective
Re. 30,985 may be used as culture media for the host cells. amount of antibody present in the formulation is determined
Any of these media may be Supplemented as necessary with by taking into account the desired dose Volumes and mode
hormones and/or other growth factors (such as insulin, (s) of administration, for example. From about 0.1 mg/mL to
about 50 mg/mL, preferably from about 0.5 mg/mL to about
transferrin, or epidermal growth factor), Salts (Such as 15 25 mg/mL and most preferably from about 2 mg/mL to about
Sodium chloride, calcium, magnesium, and phosphate), 10 mg/mL is an exemplary antibody concentration in the
buffers (such as HEPES), nucleotides (such as adenosine and formulabon.
thymidine), antibiotics (such as GENTAMYCINTM drug), An aqueous formulation is prepared comprising the anti
trace elements (defined as inorganic compounds usually body in a pH-buffered solution The buffer of this invention
present at final concentrations in the micromolar range), and has a pH in the range from about 4.5 to about 6.0, preferably
glucose or an equivalent energy Source. Any other necessary from about 4.8 to about 5.5, and most preferably has a pH
Supplements may also be included at appropriate concen of about 5.0. Examples of buffers that will control the pH
trations that would be known to those skilled in the art. The
culture conditions, Such as temperature, pH, and the like, are within this range include acetate (e.g. Sodium acetate),
those previously used with the host cell selected for Succinate (Such as Sodium Succinate), gluconate, histidine,
expression, and will be apparent to the ordinarily skilled
25 citrate and other organic acid buffers. The buffer concentra
artisan. tion can be from about I mM to about 50 mM, preferably
from about 5 mM to about 30 mM, depending, for example,
When using recombinant techniques, the antibody can be on the buffer and the desired isotonicity of the formulation.
produced intracellularly, in the periplasmic space, or directly The preferred buffer is sodium acetate (about 10 mM), pH
secreted into the medium. If the antibody is produced 5.O.
intracellularly, as a first Step, the particulate debris, either An polyol, which acts as a tonicifier and may Stabilize the
host cells or lysed cells, is removed, for example, by antibody, is included in the formulation. In preferred
centrifugation or ultrafiltration. Where the antibody is embodiments, the formulation does not contain a tonicifying
Secreted into the medium, Supernatants from Such expres amount of a Salt Such as Sodium chloride, as this may cause
Sion Systems are generally first concentrated using a com 35 the antibody to precipitate and/or may result in oxidation at
mercially available protein concentration filter, for example, low pH. In preferred embodiments, the polyol is a nonre
an Amicon or Millipore Pellicon ultrafiltration unit. A pro ducing Sugar, Such as Sucrose or trehalose. The polyol is
tease inhibitor such as PMSF may be included in any of the added to the formulation in an amount which may vary with
foregoing Steps to inhibit proteolysis and antibiotics may be respect to the desired isotonicity of the formulation. Pref
included to prevent the growth of adventitious contaminants. 40 erably the aqueous formulation is isotonic, in which case
The antibody composition prepared from the cells can be Suitable concentrations of the polyol in the formulation are
purified using, for example, hydroxylapatite in the range from about 1% to about 15% w/v., preferably in
chromatography, gel electrophoresis, dialysis, and affinity the range from about 2% to about 10% whv, for example.
chromatography, with affinity chromatography being the However, hypertonic or hypotonic formulations may also be
preferred purification technique. The Suitability of protein A 45 suitable. The amount of polyol added may also alter with
as an affinity ligand depends on the Species and isotype of respect to the molecular weight of the polyol. For example,
any immunoglobulin Fc domain that is present in the anti a lower amount of a monosaccharide (e.g. mannitol) may be
body. Protein A can be used to purify antibodies that are added, compared to a disaccharide (Such as trehalose).
based on human Y1, Y2, or Y4 heavy chains (Lindmarket al., A Surfactant is also added to the antibody formulation.
J. Immunol. Meth. 62:1-13 (1983)). Protein G is recom 50 Exemplary Surfactants include nonionic Surfactants Such as
mended for all mouse isotypes and for human Y3 (GuSS et al., polySorbates (e.g. polySorbates 20, 80 etc) or poloxamers
EMBO J. 5:1567–1575 (1986)). The matrix to which the (e.g. poloxamer 188). The amount of Surfactant added is
affinity ligand is attached is most often agarose, but other Such that it reduces aggregation of the formulated antibody
matrices are available. Mechanically stable matrices Such as and/or minimizes the formation of particulates in the for
controlled pore glass or poly(styrenedivinyl)benzene allow 55 mulation and/or reduces adsorption. For example, the Sur
for faster flow rates and shorter processing times than can be factant may be present in the formulation in an amount from
achieved with agarose. Where the antibody comprises a C3 about 0.001% to about 0.5%, preferably from about 0.005%
domain, the Bakerbond ABXTM resin (J. T. Baker, to about 0.2% and most preferably from about 0.01% to
Phillipsburg, N.J.) is useful for purification. Other tech about 0.1%.
niques for protein purification Such as fractionation on an 60 In one embodiment, the formulation contains the above
ion-exchange column, ethanol precipitation, Reverse Phase identified agents (i.e. antibody, buffer, polyol and Surfactant)
HPLC, chromatography on Silica, chromatography on hep and is essentially free of one or more preservatives, Such as
arin SEPHAROSETTM chromatography on an anion or benzyl alcohol, phenol, m-creSol, chlorobutanol and benze
cation exchange resin (Such as a polyaspartic acid column), thonium Cl. In another embodiment, a preservative may be
chromatofocusing, SDS-PAGE, and ammonium sulfate pre 65 included in the formulation, particularly where the formu
cipiation are also available depending on the antibody to be lation is a multdose formulation. The concentration of
recovered. preservative may be in the range from about 0.1% to about
US 6, 171586 B1
23 24
2%, most preferably from about 0.5% to about 1%. One or (including ischemic and hemorrhagic stroke), and myocar
more other pharmaceutically acceptable carriers, excipients dial infarction. Where the antibody is an antlL8 antibody, the
or stabilizers such as those described in Remington's Phar disorder may be an inflammatory disorder Such as adult
maceutical Sciences 16th edition, OSol, A. Ed. (1980) may respiratory distress Syndrome (ARDS), hypovolemic shock,
be included in the formulation provided that they do not 5 ulcerative colits, and rheumatoid arthritis.
adversely affect the desired characteristics of the formula E. Articles of Manufacture
tion. Acceptable carriers, excipients or Stabilizers are non In another embodiment of the invention, an article of
toxic to recipients at the dosages and concentrations manufacture is provided comprising a container which holds
employed and include; additional buffering agents; the aqueous pharmaceutical formulation of the present
co-Solvents, antoxidants including ascorbic acid and invention and optionally provides instructions for its use.
methionine, chelating agents Such as EDTA, metal com Suitable containers include, for example, bottles, vials and
plexes (e.g. Zn-protein complexes); biodegradable polymers Syringes. The container may be formed from a variety of
Such as polyesters, and/or Salt-forming counterions Such as materials. Such as glass or plastic. An exemplary container is
Sodium. a 3-20 cc single use glass vial. Alternatively, for a multidose
The formulation herein may also contain more than one 15 formulation, the container may be 3-100 cc glass vial. The
protein as necessary for the particular indication being container holds the formulation and the label on, or associ
treated, preferably those with complementary activities that ated with, the container may indicate directions for use. The
do not adversely affect the other protein. For example, where article of manufacture may further include other materials
the antibody is anti-CD18, it may be desirable to provide a desirable from a commercial and user Standpoint, including
further anti-adhesion antibody, such as an anti-ICAM-1 or other buffers, diluents, filters, needles, Syringes, and package
anti-CD11a antibody along with the anti-CD18 antibody in inserts with instructions for use.
a single formulation. Alternatively, the anti-CD18 antibody The invention will be more fully understood by reference
may be combined with another anbinflammatory agent or a to the following examples. They should not, however, be
thrombolytic agent. Such proteins are Suitably present in construed as limiting the Scope of the invention. All litera
combination in amounts that are effective for the purpose 25 ture and patent citations are incorporated herein by refer
intended. CCC.
The formulations to be used for in vivo administration
EXAMPLE 1.
must be sterile. This is readily accomplished by filtrabon
through Sterile filtration membranes, prior to, or following, This example describes an aqueous formulation compris
preparation of the formulation. ing the antibody, recombinant humanized anti-CD18 anti
D. Administration of the Formulation body (rhuMAb CD18). RhuMAb CD18 having the amino
The formulation is administered to a mammal in need of acid sequence shown in FIG. 1A (heavy chain; SEQ ID
treatment with the antibody, preferably a human, in accord NO:1) and FIG. 1B (light chain; SEQ ID NO:2) was created
with known methods, Such as intravenous administration as by humanization of the murine monoclonal antibody
a bolus or by continuous infusion over a period of time, by 35 muMAb H52 (Hildreth et al. J. Immunology 134:3272–3280
intramuscular, intraperitoneal, intrace robroSpinal, (1985)).
Subcutaneous, intra-articular, intrasynovial, intrathecal, oral, The rhuMAb CD18 was produced recombinantly as
topical, or inhalation routes. In preferred embodiments, the described below. Plasmid pS 1130 was constructed to direct
formulation is administered to the mammal by intravenous production of a rhuMAb CD18 precursor molecule with a
administration. For Such purposes, the formulation may be 40 leucine Zipper domain in E. coli. The precursor is cleaved
injected using a Syringe or via an IV line, for example. during the purification process by the protease pepsin to
The appropriate dosage (“therapeutically effective yield rhuMAb CD18. rhuMAb CD18 is an F(ab') molecule
amount”) of the antibody will depend, for example, on the composed of two different peptides (light and heavy chains)
condition to be treated, the Severity and course of the linked by disulfide bonds. Fusion of a yeast GCN4 leucine
condition, whether the antibody is administered for preven 45 Zipper dimerizabon domain to the C-terminus of an Fab'
tive or therapeutic purposes, previous therapy, the patient's substitutes for the Fc region and allows for efficient F(ab')
clinical history and response to the antibody, the type of production in E. coli. The GCN4 leucine Zipper domains
antibody used, and the discretion of the attending physician. interact to form stable dimeric structures (parallel coiled
The antibody is suitably administered to the patent at one coils) that hold the hinge region cysteine residues of two
time or over a Series of treatments and may be administered 50 heavy chains together So that the two native interchain
to the patent at any time from diagnosis onwards. The disulfide bonds can form. This results in formation of F(ab')
antibody may be administered as the Sole treatment or in complexes that are covalently linked by disulfide bonds. The
conjunction with other drugs or therapies useful in treating leucine Zipper domains are later removed from the rhuMAb
the condition in question. CD18 precursor during the purification proceSS using the
As a general proposition, the therapeutically effective 55 protease pepsin, which cleaves uniformly between the two
amount of the antibody administered will be in the range of leucine residues of the hinge. This results in the formation of
about 0.1 to about 50 mg/kg of patent body weight whether the rhuMAb CD18 F(ab') molecule.
by one or more administrations, with the typical range of Plasmid pS1130 (FIG. 20) is based on the well charac
antibody used being about 0.3 to about 20 mg/kg, more terized plasmid p3R322 with a 2143 bp expression cassette
preferably about 0.3 to about 15 mg/kg, administered daily, 60 (FIGS. 21A and 21B) inserted into the EcoRI restriction site.
for example. However, other dosage regimens may be Plasmid pS1130 is resistant to both tetracycline and
useful. The progreSS of this therapy is easily monitored by B-lactam antibiotics. The expression cassette contains a
conventional techniques. Single copy of each gene linked in tandem. Transcription of
In the case of an anti-CD18 antibody, a therapeutically each gene into a single dicistronic mRNA is directed by the
effective amount of the antibody may be administered to 65 E. coli phoA promoter (Chang et al. Gene 44:121-125
treat inflammatory disorderS Such as hemorrhagic shock, (1986)) and ends at the phage lamda to terminator
thermal injury (Such as that resulting from burns), Stroke (Scholtissek and Grosse Nucleic Acids Research 15:3185
US 6, 171586 B1
25 26
(1987)). Translation initiation signals for each chain are the pepsin-CPG column was filtered directly in line through
provided by E. coli STII (heat stable enterotoxin) (Picken et an anion exchange Sartobind Q filter (Sartorius, Goettingen,
al. Infection and Immunity 42:269-275 (1983)) Shine West Germany). The generated anti-CD18 F(ab') antibody
Dalgarno Sequences. Translation of each chain begins with flows through the filter while pepsin and other negatively
a 23 residue STI signal peptide that directs translocaton of 5 charge impurities bind Strongly to the filter. The pool was
the pepbdes acroSS the cytoplasmic membrane into the diluted to give a conductivity of approx. 7 mohms by the
periplasmic space. The STII Signal peptide is then removed addition of water and was then applied to a cation exchange
by the E. coli leader peptidase. The light and heavy chains column (SP Sepharose HP) equilibrated was 25 mM MES,
fold into their native conformations after secretion into the
periplasm and associate into the rhuMAb CD18 precursor, a 60 mM acetic acid, pH 4.0. The SP Sepharose column was
covalently linked F(ab'). The leucine Zipper domain is washed with 25 mN MES, 75 MM sodium acetate pH 5.6
cleaved from the precursor during the purification proceSS and eluted in a linear gradient of 75-110 mM sodium acetate
(see below) to yield rhuMAb CD18. The cell line used in the in 25 mN MES pH 5.6. The pooled fraction from the SP
production of rhuMAb CDI8 is 49A5, derived from E. coli Sepharose column was diluted with the addition of 3.0M
cell line W3110 (ATCC 27,325) as shown in FIG. 22. The 15 ammonium Sulphate, 25 mMMES pH 6.0 at a ratio of 0.26
fermentation procedure takes place as shown in FIG. 23. liters per liter of pool and was then passed through a
Production of rhuMAb CD18 precursor occurs when the hydrophobic interaction chromatography (HIC) column
medium becomes depleted in phosphate, typically 30-60 (phenyl Sepharose FF-low Substitution) previously equili
hours after inoculation. brated in 0.625 Mammonium sulphate, 25 nM MES pH 6.0.
Purification of rhuMAb CD18 precursor from the E. coli After loading, the column was washed with Same buffer used
cell paste was as follows. Frozen cell pellets containing in equilibration and the rhuMAb CD18 was eluted in
antCD18 precursor antibody, were dissolved in about 3 0.375M ammonium sulphate, 25 mN MES pH 6.0.
volumes of extraction buffer (120 mM MES, 5mM EDTA This resulted in purified rhuMAb CD18 for formulating as
buffer, pH 6) heated to 30-40°C. This resulted in a suspen 25
described below. Five aqueous formulations were evaluated
sion with a pH between about 5.4 and 6.5. This suspension for Selection of a clinical formulation for use in, e.g.,
was passed twice through a Gaulin homogenizer at 5500 to hemorrhagic Shock. The formulations were as shown in
6500 psi and kept below 20 C. with a heat exchanger. 5% Table 1 below.
TABLE 1.
Matrix of formulations prepared for this study.
TWEEN 20 TM NaCl Mannitol Trehalose
Formulation Buffer pH (0.01% v?v) (140 mM) (4% w/v) (8% w/v)
10 mM 5.0 -- --
Acetate
10 mM 5.0 -- --
Acetate
10 mM 6.O -- --
Histidine
10 mM 6.O -- --
Histidine
10 mM 6.O -- --
Histidine
45
polyethyleineimine (PEI) (wh), pH 6 was added to the Two pH's were compared, pH 5 (sodium acetate) and pH
homogenate to a final concentration of 0.2% PEI. The 6 (histidine). At each pH, sodium chloride and trehalose
mixture was incubated for about one hour at 2-8 C. About were compared as tonicifiers. A histidine formulation with
one volume of extraction buffer (120 mM MES, 5 mM mannitol at pH 6 was also evaluated. The presence of a
EDTA, pH 6) was added before the solids were removed by 50 surfactant (e.g. 0.01% TWEEN 20TM) was found to be
centrifugation at 15,280 g. The clear Supernatant was con required for Stabilization against Shaker-induced aggrega
ditioned to a conductivity of less than 3mohms by the tion and for preventing adsorption to containers at protein
addition of cold water. The conditioned Supernatant was concentrations below 1 mg/mL. The Stability assays indi
loaded onto a cation exchange column (ABX column; cated that the trehalose formulations were generally Superior
Mallinckrodt Baker, Inc., N.J., USA) equilibrated in 50 mM 55 to the others, with pH 5 showing slightly greater chemical
MES, pH 6.0. Sodium citrate, pH 6.0. The cation exchange Stability than the pH 6 formulation due to reduced clipping.
anti-CD18 precursor antibody pool was diluted with 50 mM Kinetic data after 2 weeks and 5 weekS Storage at 5, 30, and
MES, 36 mM sodium citrate, pH 4.0 to a concentration of 40° C. were used for Arrhenius analysis and shelf-life
approximately 2 g/L. The pool was then adjusted to pH4 by
addition of 2 M citric acid and flowed through a column prediction. Using cation eXchange HPLC, the preferred
containing immobilized pepsin (pepsin-CPG) previously 60 formulation for rhuMAb CD18 was 10 mM Sodium acetate,
equilibrated with 50 mM MES, 36 mM sodium citrate pH 8% trehalose whv, 0.01% TWEEN 20TM, pH 5.0, with a
4.0. Pepsin (Sigma, Mo., USA) was chemically coupled to predicted shelf life of 25-75 months (95% confidence
controlled pore glass (CPG) by Bioprocess Ltd., NJ, USA; intervals) at 5° C.
the CPG was activated with NalO4 followed by reduction of In designing antibody formulations, it may be useful to
Schiff base formation between CPG and pepsin using 65 analyze the Structural properties of the antibody to be
NaBH3CN). This procedure removed the zippers from the formulated, but this is not necessary. RhuMAb CD18 con
hinge region while leaving intact F(ab'). The effluent from sists of beta sheets of light (left side of the molecule in FIG.
US 6, 171586 B1
27 28
16) and heavy chains (right side of the molecule in FIG. 16) Final pH 5.92. The solution was sterile filtered into 2 L
which are structurally stabilized each by two intramolecular Nalgene bottles and stored at 5 C.
disulfides and held together by two intermolecular disulfides 2 M NaCl: 116.88 g NaCl, made up to 1 L with Milli-Q
(depicted as barrels at the bottom of FIG. 16). The CDRs water. The solution wasd sterile filtered into 1 LNalgene
(complimentarity determining regions) in the variable Seg 5
ments are oriented on the top of the molecule. Located bottles and stored at 5 C.
within and close to the CDRS are a number of methionines, 18% Mannitol: 180 g mannitol, made up to 1 L with
only one of which (Met 65) is fully exposed. Though Met 65 MilliO water. The Solution was sterile filtered into 1 L
is buried in anti-CD18, it is near a histidine residue which, Nalgene bottles and stored at 5 C.
Without being limited to any one theory, might promote 10 20% Trehalose: 400 g trehalose, made up to 2 L with
metal induced oxidation of proximal methionine (Li et al. Milli-Q water. Final pH was 6.4. The solution was
Biochem. 34(17):5762-5772 (1995)). Susceptibility to oxi- sterile filtered into 2 L Nalgene bottles and stored at 5
dation in the CDRS was assessed herein. It was found to be C
desirable to use a Sugar as the tonicifler, rather than a Salt, So
as to minimize oxidation at low pH. 15 10% (v/v) TWEEN 20TM: 10 mL of concentrated TWEEN
In assessing the potential deamidation or isomerization 20TM was carefully removed and added to a 100 mL
Sites in this molecule, i.e. ASX-Gly or ASX-Ser (ASX indicates volumetric flask, diluted with Milli-Q water, and stirred
Asp or ASn) that are in hydrophilic and flexible regions until dissolved. Stored at 2-8 C. in the dark.
(Clarke et al. in Stability of Protein Pharmaceuticals. Part Formulation: Table 2 below shows the preparation of the
A. Chemical and Physical Pathways of Protein 2 buffers against which the starting protein bulk was dialyzed
Degradation, T.J. Ahern and M. C. Manning, Editors. 1992, for this study. Approximately 40 mL of the bulk rhuMAb
Plenum Press: New York. p.2-29; and Kossiakoff, Science, CD18 (~1.3 mg/mL protein concentration) was dialyzed (2
240:191-94 (1988)), six were found to be exposed and in L for 4 hr at 5° C. and ~2 Lovernight) to solutions prepared
fairly flexible regions (FIGS. 2A and 2B). Without being as shown in Table 2. The dialyzate was filtered with 0.2 uM
limited to any one theory, heavy chain motifs MNS, Nalgene cellulose acetate filter units and Stored under asep
KNG, and light chain motifs GN'S, QD’S, KD'7°S, tic conditions for use as blank for UV and HPLC analysis.
TABLE 2
Summary of formulation preparation
O.2 M 18% 20% Approx. Made up
buffer Mannitol Trehalose 1M NaCl Water tO
Formulation (mL) (mL) (mL) (mL) (mL) pH final pH (mL)
F1 Acet/5 2OO 560 3OOO 5.07 5.07 4OOO
F2 Acet/5 2OO 16OO 2OOO 5.17* 5.07 4OOO
F3 His/6 2OO 560 3900 6.03 6.03 4OOO
F4 His/6 2OO 889 28OO 5.85* 6.O1 4OOO
F5 His/6 2OO 16OO 2OOO 5.97 5.97 4OOO
Formulation F2 was adjusted with acetic acid buffer made by diluting 286 uL of glacial acetic
acid and 200 mL of the 20% trehalose and mixed with Milli-Q water to a final 500 mL. This is 10
mM acetic acid with 8% trehalose. Approximately 250 mL was required to adjust the pH as
required. Formulation F4 was adjusted with 0.5 mL of 50% NaOH.
are predicted to be most reactive, reactivity at ASn 84 is most 4

Some of the remaining dialysis buffer was used to prepare
likely to affect the binding activity of the antibody since it formulation vehicles by adding TWEEN 20TM, as shown in
is near the CDR's. ASn 103 was found to be the primary Table 3.
route of degradation (via deamidation).
MATERIALS AND METHODS TABLE 3
50
The materials used in the following methods were as Summary of preparation of formulation vehicles
follows: glacial acetic acid 99.9% (MW 60.05); concen
trated NaOH 18.94N (50% w/w; MW 4.0.0); concentrated Dialysis 10% Final
HCI 37.8% (12.44N; MW 36.46); hisbidine (MW 155.16);
NaCl (MW 58.44g); pharmaceutical grade trehalose dihy 55
Buffer TWEEN 20 TM Vol.
drate (MW 342.31); D-mannitol (MW 182.17); low perox Vehicle (mL) (uL) (mL)
ide TWEEN 20TM; rhuMAb anti-CD18 (~1.3 mg/mL in
MOPS, Na acetate, pH 6.9). F1 41.4 41 41.4
Buffer preparation and formulation set-up: Two liters of F2 41.5 42 41.5
the following Stock buffers were prepared for dialysis and 60
F3 42.4 42 42.4
formulation:
F4 42.2 42 42.2
0.2 M Sodium Acetate, pH 5:22.7 mL acetic acid, 17 mL
50% NaOH, made up to 2 L with Milli-Q water. Final F5 41.4 41 41.4
pH 4.98. The solution was sterile filtered into 2 L
Nalgene bottles and stored at 5 C. 65
0.2 M Histidine, pH 6: 62.06 g of Histidine, 20 mL Table 4 below shows the final appearance, pH, and protein
concentrated HCI, made up to 2 Lin a Volumetric flask. concentration of the dialyzed protein Solutions.
US 6, 171586 B1
29 30
TABLE 4
Summary of analysis of the dialyzed protein solutions
OD28O
diluted Conc. = pH of protein colorf Temp
Formulation 1:1 OD/1.32 solution Vehicle pH appearance C.
F1 O.8239 1.248 5.09 5.09 CAC* * 24.7
F2 11192. 1.696 5.09 5.09 CAC 24.7
F3 O.84605 1.282 6.06 6.06 CAC 24.7
F4 O.89763 1.36 6.10 6.10 CAC 24.7
F5 1116 1.69 5.91 5.91 CAC 24.7
*Extinction coefficient was later changed to 1.45 mL/mg/cm.

**CAC = clear and colorless.
15
These protein Solutions were then diluted, using reserved

dialysis buffer, to a final ~1 mg/mL protein concentration,
and TWEEN 20TM was added to final 0.01% (v/v), as shown
in Table 5.
TABLE 5
Summary of final formulation concentration adjustment
and concentration determination
Dialysis 10% Dialy. Starting Final *Final

Form. buffer TWEEN2O TM Protein Conc. Vol. Final Conc. *OD
O. (mL) (uL) (mL) (mg/mL) (mL) OD280 (mg/mL) 340-360
F1 8.16 41 33.2 1.248 41.4 13.297 1.008 O.O11
F2 17.0 42 24.5 1.696 41.6 13332 1.010 O.O17
F3 9.26 42 33.1 1.282 42.4 13138 O.995 O.O09
F4 11.2 42 31 1.36 42.2 1.3065 O.990 O.O10
F5 16.9 41 24.5 1.69 41.4 13118 O.994 O.O16
* Measured after filtration and filling.

35
The formulations were sterile filtered using 150 mL TABLE 6B

Nalgene filter units with cellulose acetate membranes in a Vehic
CCIeS
sterile hood. The sterile formulated rhuMAb CD18Solutions 40
were filled at 0.6 mL per 3 cc vial, labeled and stored at time
40 C.
vehicles
30 C.
vehicles
5 C.
vehicles
–20 C.
vehicles
–70 C.
vehicles
designated temperatures. Vehicle Solutions were filtered and
filled identically. Tables 6A (antibody formulations) and 6B 2 tO
wk 1. 1. 1.1. 1. 1.
(vehicle controls) below show the stability program for these as 5 wk
8 wk
1.
1.
1.
1.
1.
1.
1. 2
formulations. Data presented here are up to 5 weekS Storage. 12 wk 1. 1. 1. 1. 2
6 mo 1. 1. 1. 2
TABLE 6A 1 yr 1. 1. 1. 2
4 6 6- 2=8 4+2 =8 8 + 2 ext
50
CD18 antibody formulations
At the 2 week and 5 week timepoints, 2 vials from each
formulation and Storage temperature were frozen at -70 c.
40° C. 30° C. 5° C. -20° C. -70° C. At the 5 week timepoint the frozen Samples were thawed and
time samples samples samples samples samples batch analyzed together with control Samples froZSen at
55 -70° C. immediately after filling (t=0 weeks).
tO 2 2 2 UV Spectroscopy: Samples were measured without dilu
2 wk 2 2 2
tion in a Hellma 2 mm wide, 1 cm path-length cell with
raised bottom and black sides. Approximately 300 till of
5 wk 2 2 2 2 2
Sample was used for the measurements. The instrument was
8 wk 2 2 2 60 blanked with Milli-Q water. Samples were scanned from
12 wk 2 2 2 2 2 200 to 400 nm with a 2 nm bandwidth and 1 second
6 mo 2 2 2 2 integration using a HP-8452A spectrophotometer.
Protein concentration was determined from the absor
9 mo–1 yr 2 2 2 2
8 + 2 ext= 10 12 14 8 10 + 4 ext= 14 65
bance at 278 nm correcting for the absorbance at 320 nm
using an extinction coefficient of 1.45 mL/mg/cm. The
average Value of absorbances in the region of Aso to Asoo
was followed as an indicator of Scattering material.
US 6, 171586 B1
31
pH: An Orion 720A pH meter was used with a
MicroElectrodes, Inc. MI-410 micro pH electrode. The TABLE 9
electrode was calibrated with pH 4 and 7 standard buffers, Time %B
re-checked every 10–15 Samples, and recalibrated as
O 1O
required. The instrument was calibrated and Samples were 1. 1O
measured at room temperature (~23 C.). 35 1OO
RP-HPLC: RP-HPLC was carried out using a 7.5x75 mm 37
37.1
1OO
1O
TSK Phenyl-5PW column with 0.1% TFA as the A buffer, 42 1O
and 0.08% TFA in acetonitrile as the B buffer. An inline 1O
0.5.M filter was used before the column was equilibrated at Data were collected at 214 nm at 1 point per second.
10% B buffer and run at 55° C. Injections of 20 uL were SDS-PAGE: Pre-made 10 and 14% glycine SDS-PAGE
made and eluted according to Table 7 below: gels (Novex, San Diego, Calif.) were used for analysis of the
reduced and non-reduced Samples, respectively. 10 till of the
TABLE 7 15 1 mg/mL sample diluted with 10 till of 2xTris-Glycine
Time Flow
sample buffer (Novex) was heated 2 minutes at 95 C., as
(Minutes) %B (mL/minute)
were high MW range and low MW range (Bio-Rad) markers
diluted 1:20 in same sample buffer. Kaleidoscope MW
O 1O O.8 marker was heated at 95 C., as well, with no dilution. When
3O 25 O.8 the Samples cooled, 10 till was loaded on the gels. The load
35 70 O.8 of the MW marker Solutions was 5 ul. The gel apparatus
35.01 70 2
36 70 2 (Novex) was filled with 1xgel loading buffer (Media
37 1O 2 Services) and the gels were run at constant voltage of 125
41 1O 2 mV and variable current for 2 hours. The gels were stained
Data collected at 214 nm at 1 point per second were used for analysis.
25 with the Novex Coomassie blue stain, overnight. The next
day, the gels were washed with water for 2 hours and then
SEC Assay: The mobike phase used was 200 mM NaCl, were soaked in Gel Drying Solution (1x) (Novex) for '/3
50 mM sodium phosphate at pH 6.0. The runs were 40 hour. The gels were then air-dried with the Novex cello
phane System.
minutes at a flowrate of 0.5 mL/minute with 40.u.L injectons Gel Isoelectric Focusing Electrophoresis: IEF was done
done at ambient temperature. A TSK G3000SWXL 30 using Pharmacia 3.5 to 9.5 PAG plate gels. Pharmacia
cmx7.8 mm column with inline filter and a TSK guard phosphoric acid and Sodium hydroxide running buffers were
column before the main column was used. Detection was at used. Samples were prepared by mixing 10 till of Sample
214 nm. with 30 ul of 15% glycerol. Pharmacia broad range and high
Ion-Exchange HPLCASsay(IEX): IEX was run using 50 35 range IEF standards, as well as Serva Protein Test Mix 9
till injections. The mobile phases were 33 mM each of were run as pi markers. Pharmacia Standards were prepared
MES/HEPESIPIPES, adjusted to pH 6.0 for A and pH 8.0 by mixing the standard (reconstituted with 0.5 mL of
for B; C=Milli-Q water. The gradient shown in Table 8 Milli-Q water) 1:1 with 15% glycerol. The Serva protein
below was used: mix was reconstituted according to instructions and diluted
40 10-fold with 15% glycerol. On one gel, anti-HER2 MAb
TABLE 8
bulk (pl=8.8-9.0 calculated) was also run as a control. The
HER2 sample (5 mg/mL) was diluted 40x with 15% glyc
Time Flow erol. The gels were loaded with 20 u, of each sample
(min) %B %C (mL/min) adsorbed onto paper patches placed one third of the gel
O O 50 1.
45 width from the positive electrode (the acid side). Gels were
3 O 50 1. run at 10 C. with constant voltage set to 400 V for 30
15 2O 50 1. minutes, after which the Sample loading papers were
50
SO.1
50
1OO
50
O
1.
2
removed and the gels were run for 1.5 more hours at 1500
51 1OO O 2
V. Gels were fixed in TCA with sulfosalicylic acid for 30
51.1 O 50 2
50 minutes, and washed with water for 10 minutes (for the
60 O 50 2 Second gel) or IEF destain (for the third gel), and stained
without washing out the TCA, as instructed in the Novex
instructions (for the first gel). The first two gels were stained
The method used a 50x4.6 mm BakerBond carboxy with Novex colliodal Coomassie according to the Novex
Sulphon (CSX) cation exchange column with an inline 0.5 55 instructions. The last gel was Stained for 8 minutes with
luM filter before the column, run at 40 C. Analysis was done standard IEF Coomassie stain which was preheated to ~60
with data obtained at 280 nm, due to the high buffer C., and then destained with IEF destain which had also been
background. preheated to ~60° C. overnight. The gels were air-dried with
Hydrophobic Interaction Chromatography (HIC): HIC the method described above for the SDS-PAGE gels.
was done on a 4.6x50mm Baker Bond Butyl NPR column
60 Freeze/ThawStability: To demonstrate that rhuMAb
CD18 would not degrade by freeze/thaw cycling in the
with inline 0.5 uM filter before the column. The running formulations being tested, four vials of each formulation
buffers were 2Mammonium sulfate in 20 mM Tris-HCI, pH were Subjected to three cycles of freezing and thawing. Two
7 as the Abuffer, and 20 mM Tris, pH 7 as the B buffer. The of the vials of each formulation were cycled between -70
column was equilibrated at 50° C. with 10% buffer B at 1 65 C. and 2-8 C. and two were cycled between -20° C. and
mUminute and 10 till injections were made. The gradient 2-8 C. Two vials of each formulation were held at 2-8 C.
shown in Table 9 below was used for elution: as controls. During each cycle the Vials were allowed to
US 6, 171586 B1
33
remain in the freezer for at least 2 hours, until there was no
Visible liquid remaining in the vial. They were then trans TABLE 10
ferred to 2-8 C. for at least 2 hours, until no solid remained.
One vial of the acetate/trehalose, pH 5 formulation (F2) did Dilution Sample Diluent
not freeze at -20°C. on the first cycle, but froze immediately Step (times) (uL) (uL)
when placed on the shelf in a -70° C. freezer. 1, in 96 well plate* 1O 1O 90
Shaker Studies: One vial of each formulation, and a vial 2, in 96 well plate* 1O 1O 90
of identically configured rhuMAb CD18 bulk, was placed 3, in 96 well plate* 1O 1O 90
horizontally into a rack on a Glas-Col 99A S60012 shaker. 4, in Micronics tubes 2O 50 950
5, in Micronics tubes 2 500 500
The shaker arm was adjusted to a radius of ~30 cm and the 1O 6, in Micronics tubes 2 500 500
speed was set to 70 rpm. The samples were shaken for 24
hours at room temperature in the axial direction. A Second *Later studies were conducted by making all dilutions in Micronics tubes
Vial of each formulation, and the bulk, was maintained at and vortexing them to mix; this improved reproducibility.
room temperature near the Shaker, as a control. The Samples Kinetic Parameters, Arrhenius Calculations, and Statis
were then analyzed by IEX, MAC-1 capture assay, and UV 15 tics: The loss in the % main peak versus time was fitted to
SpectroScopy.
MAC-1 Capture (Receptor ELISA) and Total F(ab') first order kinetics. The observed rate constants (K) at
ELISA: rhuMAb CD18 is directed to the CD18 chain of
various temperatures were then fitted to the Arrhenius equa
tion as shown below:
MAC-1. The antigen binding property of rhuMAb CD18 can
hence be evaluated by “MAC-1 capture assay”. The assay In K=-AEART-C
involves coating of a 96 well mictrotitre plate first with a
murine anti-CD18 antibody that binds to a site of MAC-1 where E is the activation energy (in cal/mol), R is the
different from rhuMAb CD18 (MHM23 MAb; Hildreth etal, Universal gas constant (1.987 cal/mol/K), T is the absolute
Eur: J. Immunol. 13:202-208 (1983)) to capture a recombi temperature (in Kelvins) and C is a constant. From the slope
nant preparation of soluble MAC-1, followed by a wash with 25
of a plot of In KVS. 1/T, AE was calculated. The standard
phosphate buffered saline (PBS), addition of the unknown error (SE) of In K was calculated using the formula
sample, wash with PBS, addition of goat HRP-labeled (1/K)+(SE). The reason for a large SE value for In K
anti-human F(ab') antibody and colorimetric detection of at lower temperatures is the Small value of the K which is in
OPD substrate. The total amount of antibody is measured by the denominator of the formula. A value of K extrapolated
first coating an ELISA plate with a polyclonal anti-F(ab') from the Arrhenius fit (+95% confidence interval) was used
antibody, followed by addition of the sample, and then to predict the shelf life at 5 C. according to:
HRP-anti F(ab') and calorimetric detection of the HRP
substrate OPD. Assay diluents in both cases were 0.5%
bovine serum albumin (BSA), 0.05% polysorbate 20, 1 mM where K was expressed in weeks'. Confidence intervals
CaCl, 1 mM MgCl2 Specific activity was measured as the 35 were determined using the program SIGMAPLOTTM.
ratio of the MAC-1 binding to total F(ab'), ELISA value. RESULTS AND DISCUSSION
The Samples were Serially pre-diluted with assay diluent
obtained to final dilutions of 20,000, 40,000, and 80,000 Freeze/Thaw stability: Table 11 below shows a Summary
times in microtiter plates and MicronicS tubes using multi of the data from analysis of the Samples in the freeze/thaw
channel pipettors. The dilution Scheme used was as shown in 40 Study. No Significant differences were Seen between the
Table 10 below. Samples of diluted rhuMAb CD18 bulk different temperature groups. The observation that one vial
were included on each assay plate as controls. These bulk of F2 did not freeze at -20° C. on the first cycle raised a
controls were diluted one extra 2xdilution to final 40,000, concern that freezing of bulk in a jacketed StainleSS Steel
80,000, and 160,000 times. The same dilutions were used for Storage tank might be problematic. The coldest parts of the
both assayS. Samples were diluted the same day as they were 45 solution in these tanks reach only about -20° C., and
thawed for the 5 week analysis and then frozen in the therefore might not freeze completely. This was tested using
MicronicS tubes prior to Submission for assay the following formulation vehicle. The tank was observed to freeze Solid
day. The freezelthaw stability of rhuMAb CD18 diluted in within 6 hours of cooling, So no problem for bulk Storage is
assay diluent in these assays was verified. predicted.
TABLE 11
Effect of three cycles of freezing and thawing to 5 C.
on the Stability of rhuMAb CD18
MAC-1
Capture
RP-HPLC RP-HPLC SEC SEC UW Conc. UV Average Titer Specific Activity
Formulation Total Area %. Main Total Area %. Main (mg/mL) 340-360 nm. (ug/mL) (MAC-1/UV)
F15° C. 10276 - 25 88.0 - 0.1 6401 - 151 83.8 O.4 O.949 - O.OO3 O.O11 O.OO1 1000 - 32 1.05 + 0.03
-2O. C. 10341 - 106 88.2 - 0.1 6556 73 83.2 - 0.1 O.952 O.OO2 O.O12 O.OO1 948 - 28 1.OOOO3
-70° C. 10328, 13 87.5 O.O1 6353 63 83.O. O.9 0.948 0.005 0.013 - 0.004 972 - 15 1.O3 + O.O2
F2 5° C. 10537 - 4 88.5 + 0.2 6508 - 18 81.7 O.3 O.941 O.OO6 O.O17 O.OOS 1069 57 1.14 OO6
-2O. C. 10644 - 107 87.3 - O.S 6290 - 54 83.1 O.2 O.941 O.OO2 O.O12 O.OO1 1132 27 1.20 O.O3
-70° C. 10503 - 74 88.5 + 0.2 6435 - 120 82.5 + 0.8 O.938 - O.OOO O.O09 OOO2 1064 41 1.13 - 0.04
F3 5° C. 10147 SO 87.9 O.2 6311 - 65 848 - O.3 O.926 - O.OO2 O.O17 O.OO1 1017 77 1.10 O.08
-2O. C. 10050 + 43 88.4 O.OO 6404 - 39 85.6 - 0.1 O.922 O.OO7 O.O12 O.OO1 997 - 28 1.08 O.O3
-70° C. 995385 88.0 O.2 6356 52 83.9 O.2 O.926 - O.OO2 0.017 - 0.005 1021 + 26 1.O. O.O3
US 6, 171586 B1
35 36
TABLE 11-continued
Effect of three cycles of freezing and thawing to 5 C.
on the Stability of rhuMAb CD18
MAC-1
Capture
RP-HPLC RP-HPLC SEC SEC UW Conc. UV Average Titer Specific Activity
Formulation Total Area %. Main Total Area %. Main (mg/mL) 340-360 nm. (ug/mL) (MAC-1/UV)
F45° C. 10397 - 26 88.2 - 0.1 6304 - 148 84.5 + 1.1 O921 - O.OOO 0.010 - 0.003 905 - 18 O.98 O.O2
-20 C. 10126 - 18O 88.6 O.O3 6467 13 83.5 + 0.2 O.922 - O.OO)4 O.O14 O.OO1 1004 20 1.09 O.O3
-7O C. 1032251 88.1 - 0.1 63.273 84.7 - 1.1 O924 - O.OO)4 O.OO9 O.OOf 982 21 1.06 O.O3
FS 5° C. 10438 81 88.0 - 0.1 6418 64 85.9 0.3 O.930 - O.OO1 0.016 - 0.004 1058 - 39 1.14 - 0.04
-20 C. 10443 - 10 88.3 - 0.1 631O 11 86.5 + 0.4 O.923 - O.OO)4 O.O16 O.OO3 1010 49 1.09: 0.05
-7O C. 1035O 135 88.3 + O.OO 6340 - 58 85.5 + 0.1 O.923 - O.OO3 O.O17 O.OO6 1003 22 1.09 O.O2
Mean + SE of two vials are shown. No significant loss in monomer content or receptor binding was noticed.
UV SpectroScopy: Spectroscopic analysis of the Samples

was done to measure protein concentration as well as to TABLE 12-continued
measure light Scattering as indicated by an increase in
average absorbances in the range of 340 to 360 nm, or by a Formulation pH after 5 weeks storage.
decrease in the ratio of the absorption maxima at 278 nm and pH of Active
the minima at 252 nm. FIGS. 3A and 3B show a summary Sample pH of Vehicle (both vials shown)
of the UV data. The 40 C. samples of formulation F3 were F3 at 5° C. 6.160 6.130/6.086
very cloudy, containing white flocculent particles. These 25
F3 at 30° C. 6.204 6.138/6.090
samples were microfuged for 5 minutes at 14000 RPM and F3 at 40° C. 6.214 6.098/6.075
re-measured after which the Scatter was still relatively high. F4 at -70° C. 5.964 6.014f6.016
All other Samples appeared clear and colorless (A. F4 at -20° C. 6.017 6.050/6.019
F4 at 5° C. 6.021 6.004/5.988
360-0.02, A-7s/As>2.4). The change in A278/A250 was a F4 at 30° C. 5.928 5.914/5.897
more Sensitive indicator of Scattering than the average F4 at 40 C. 5.88O 5.849/5.854
absorbances from 340 to 360 nm, since the latter showed no F5 at -70° C. 5.653 5.751 (5.754
storage temperature related trend (FIG. 3A), but the former F5 at -20° C. 5.729 5.761/5.766
F5 at 5° C. 5.727 5.768/5.777
showed a definite increase for the 40°C. samples (FIG.3B). F5 at 30° C. 5.797 5.843/5.833
The pH 5 formulations showed a smaller change in A278/ F5 at 40° C. 5.812 5.822/5.831
A252 ratio at 40 C. than the pH 6 formulations in every 35
case. The protein concentrations for the pH 5 formulations
were only 2-3% greater than the pH 6 formulations (FIG. 4). The pH 5 formulations showed a small increase in pH at
higher Storage temperatures for the vehicles and active
pH: All formulation vehicles except F4 showed a slight samples. Formulations F3 and F5 showed increasing pH in
increase (-0.1 units at 40°C.) in pH, compared with -70° C. 40 the vehicles with higher Storage temperature, but the active
controls (Table 12 below). Both pH 5 formulations showed Samples did not show this increase. Formulation showed a
a greater pH increase in the active Samples than in the Slight reduction in pH.
vehicles. None of the pH 6 formulations showed this dif SEC: FIGS. 5A and 5B show the chromatograms for
ference between active and vehicle Samples. In fact, the pH formulations F2 (FIG.5A) and F5 (FIG. 5B). The main peak
increase seen in the F3 and F5 vehicles was not seen in the 45 of rhuMAb CD18 eluted at 18.6 minutes, corresponding to
active samples. The pH of F4 decreased slightly at higher an apparent MW of ~70,000 D. There were two major
Storage temperature. The pH increase in the pH 5 Samples impurity peaks that eluted after the main peak, and the one
could be explained by a loSS in acetic acid concentration due at 20.9 minutes increased considerably with increasing time
to diffusion through the topper, and is not expected to be and Storage temperature. The increase in the peak at 20.9
significant at 5 C. 50 minutes was much more pronounced in F5 (FIG. 5B)
compared to F2 (FIG. 5A), without being limited to any one
TABLE 12 theory, most likely due to the higher pH of F5. FIGS. 6A and
Formulation pH after 5 weeks storage. 6B show graphs of the total peak area and the % main peak
for all of the formulations after 0, 2 and 5 weeks of storage
pH of Active 55 at 5, 30 and 40 C. The total peak areas for formulations F3
Sample pH of Vehicle (both vials shown) and F4 were significantly lower than the other three formu
F1 at -70° C. 4.892 5.009/5026 lations.
F1 at -20° C. 4.894 5.028/5.035 Since the lower pH formulations showed the greatest
F1 at 5° C. 4.947 5.07715.082 stability, rate data from the 9% main peak of the SEC
F1 at 30° C. 5.040 5.154/5.164
F1 at 40° C. 5.045 5.17115.171 60 chromatogram were calculated and fitted to an Arrhenius
F2 at -70° C. 5.037 5.17975.089 plot. Data for F2 is shown in FIG. 7. This plot contained only
F2 at -20° C. 5.041 5.096/5.101 two useful points since the rate constant at 5 C. had a large
F2 at 5° C. 5.098 5.15115.153 Standard error. The activation energy obtained from these
F2 at 30° C. 5.134 5.192/5.213
F2 at 40° C. 5.136 5.242/5.215 data (19+6 kcal/mol) was very similar to the one calculated
F3 at -70° C. 6.123 6.164f6.154 65 from the IEX data (see FIG. 15) even without the 5° C.
F3 at -20° C. 6.162 6.155/6.138 datum. The 95% confidence intervals to the curve fit predict
a shelf-life from 6 to 60 years at 5 C.
US 6, 171586 B1
37 38
HIC: This assay was performed at a column temperature
of 50° C. rather than 55 C. to improve protein recovery TABLE 13
(from -30% to nearly 90%), unfortunately at the expense of
loss in resolution. HIC analysis of the stability samples
showed an increase in a poorly resolved peak on the leading First order rate constants (K + SE; n = 2)
shoulder of the main peak, as well as an increase in a 17.5 for all formulations based on 26 main of IEX
min peak with increasing Storage time and temperature for
all formulations (FIG. 8). The shoulder was in a position that Form
would be consistent with an oxidized form of the protein, ulation 5° C. 30° C. 40° C.
though in preliminary assessment no peaks corresponding to
an oxidized Species appeared by peptide mapping. F2
F1 OOOO64 O.OO21 O.OO83 O.OO14 O.O299 O.OO23
showed the smallest increase in this leading peak (FIG. 9).
A trend towards reduced recovery (total area) was also seen F2 O.OO65 O.OO23 O.OO91 O.OO25 0.0245 + 0.0040
with increasing Storage temperatures, with all formulations F3 -OOOO4 OOOO8 O.O143 O.OO1O O.O432 O.OO19
showing the same total area at 40° C. (FIG. 10). Formula 15 F4 -OOOO66 OOOO73 O.O137 O.OOO33 O.O413 O.OOO22
tions F2 and F5 showed the least change in total area at 5 and F5 -0.00010 - 0.00082 0.0129 + 0.00095 O.O439 O.OO17
30° C. relative to the starting -70° C. sample. This method
was not Stability indicating because the resolution between
the degradation peak at 17.5' and the main peak is too low
for accurate quantitation, and the recovery from the method
is poor.
RP-HPLC: The only change observed by RP-HPLC was Formulations F1 and F2 are clearly better than the other
an increase in a peak eluting at about 25 minutes, with a formulations, implying that the major degradation process is
corresponding reduction in the main peak area, as the base catalyzed. An Arrhenius plot based on the % main peak
Storage temperature increased (FIGS. 11 and 12). The mag 25 area predicted an activation energy of 19.8+9.9 kcal/mole
nitude of this change was similar to that of the lower MW for F2 and a too of 50+25 months (95% confidence intervals)
species seen in SEC. Based on the relative position of this at 5° C. (FIG. 15). Receptor binding activity: Table 14 below
peak and its area, it may correspond to the low MW species. shows the Specific activity for each temperature and formu
Formulation F2 showed the least change in the % main peak lation after 5 weekS Storage. The Specific activity was
area with increasing temperature. Samples reduced with
DTT were also run on RP-HPLC to increase the chance that calculated by dimding the MAC-1 capture assay concentra
an oxidized form might be resolved from one of the Sepa tion by the total F(ab'), ELISA concentration. The standard
rated Smaller Subunits of the antibody. No difference was error shown is for n=2 and accounts for the total propagated
seen between the stored samples and the -70° C. control error from the two different assays for 2 vials at 3 dilutions
samples with this method. 35 each. There was a clear trend for all formulations towards a
The Arrhenius plot of the RP-HPLC kinetic data for lower Specific activity at higher Storage temperatures
formulation F2 gave a similar activation energy (21+1 (~10-15% loss at 40°C.); all formulations showed about the
kcal/mole) to the SEC and IEX results (19–20 kcal/mole) same extent of loss at 40 C. relative to ~70° C. The p value
(FIG. 13). The shelf-life indicated by the 95% confidence from one way ANOVA analysis of the 40 C. data for the
intervals to the curve fit is 4 to 150 years at 5 C. 40
different formulations is 0.98, so the 40 C. means would not
IEX: By this assay, earlier eluting peaks with retention be considered significantly different from each other, within
times of 18.4 and 26.6 minutes increased with increasing 95% confidence. An unpaired t-test of the 40 C. data versus
storage temperature. As shown in FIGS. 14A and B which
represent formulations F2 and F5, respectively, there was a the 30° C. data shows that the difference is significant
more pronounced increase of these peaks in F5, compared to 45 (p=0.044) and the lower temperatures are even more sig
F2. Also the peak at 19.5 minutes became broader at higher nificantly different by this method (p<0.02).
TABLE 1.4
Storage
Temp ( C.) cal cal lea SE
-70 O.976 O.O98 O.906 O.112 0.952 O.918 O.433

-20 0.978 O.123 O.903 O.152 O.871 O865 O.209
5 O.982 0.157 O898 O.166 O.919 O886 O.350
3O O.923 OO67 O.878 O.O76 O868 O.885 O.340
40 O.866 O.113 O.817 O. 115 O.817 O.772 O.198
temperatures. Based upon chromatographic changes Seen 60 IEF: The effect of storage for 5 weeks at different tem
upon exposure of Samples to pH 11 (see below), this method peratures was assayed by isoelectric focusing electrophore
should readily detect changes Such as deamidation and/or sis (IEF) for formulations F2 and F5. The major band
disulfide scrambling, both of which can be induced at appeared to have ap of -8.8. Three main components at pl’s
alkaline pH. ~8.75 (second most intense band), -8.6, and ~8.4 were also
65 Seen, which increase slightly with Storage, particularly at pH
Table 13 below shows the first order rate constants for the 6. The 30 and 40 C. samples showed conversion to the more
5 formulations at 5, 30, and 40° C. acidic bands. For the 40 C. samples, the bands at pl8.8 and
US 6, 171586 B1
39 40
8.75 had approximately equal intensity and there was no may have been formed either by proteolytic cleavage and/or
discernible difference between the formulations in this by disulfide scrambling, both of which may be enhanced at
regard. There were at least two more acidic bands (pl’s ~8.6 higher pH S. Reduction of the control and degraded Samples
and ~8.4), which had at least 4-fold lower intensity than the led to only the light and heavy chains, consistent with either
two most basic bands. These bands did not seem to change the cleavage to Fab, or mixed disulfide formation between
in intensity with Storage. heavy and light chains.
SDS-PAGE: The effect of storage temperature on the RhuMAb CD18 appeared to be least stable in salt formu
stability of rhuMAb CD18 formulations F2 and F5 (5 lations; at pH 5 a larger aggregate (equivalent to a trimer)
weeks) was assayed by SDS-PAGE (under reduced and appeared and at pH 6 precipitation was noted at 40 C.
nonreduced conditions). The rhuMAb CD18 appeared at a Without being bound to any one theory, one possible expla
MW of ~120 kD on the non-reduced gel. In the starting bulk nation is that NaCl does not provide hydrogen bonding, as
there were Several minor bands at higher molecular weight Sugars and mannitol do, which could potentially prevent
than the main band which may represent rhuMAb CD18 Self-association of this antibody at higher temperatures.
with various portions of the leucine-Zipper Segment Sill Based on the above preliminary data from the primary
attached. Several percent of this impurity is known to be 15
assays (IEX, SEC, and UV), the preferred aqueous formu
present in this bulk preparation. There were Several bands of lation for rhuMAb CD18 is 10 mM sodium acetate, 8%
lower MW than the main band, but only one of these bands trehalose wN, 0.01% TWEEN 20TM, pH 5.0. The shelf-life
Seemed to change in any of the formulations, or with predicted from an Arrhenius fit to the first order rate con
temperature. The band at around 45 kD increased with stants (IEX data) is 1.7 to 4 years at 5° C. (95% confidence
temperature and Seemed slightly more intense in pH 6 than intervals).This formulation can be prepared by mixing 0.573
in pH 5 formulations. This observation was consistent with mL glacial acetic acid, 0.403 mL concentration NaOH, 80 g
SEC data. The band just above the -45 kD band on the trehalose, 1 mL of a 10% TWEEN 20TM solution and making
non-reduced gels corresponded in apparent molecular up to 1 L with MilliO water (pH 5.0+0.1 at the 2 L scale).
weight with the band Seen on the reduced gels and may Conductivity of this formulation was found to be 502+10%
represent a non-reducing contaminant protein. All Species 25
microSiemenS/cm using a Radiometer-Copenhagen CDM
converted to two bands, corresponding to light and heavy 83 with a CDC 314 probe, and density was 1.017 g/mL.
chain, upon reduction with dithiothreitol, Suggesting the
absence of any proteolytic cleavage. MALDI-TOF MS EXAMPLE 2
(Yates, J. Methods Enzymol., 271:351–377 (1996)) con This example describes the production of a Stable aqueous
firmed formation of fragments that were the size of Fab'. multdose formulation comprising a recombinant humanized
Formulation pH and protein concentration: The effect of anti-CD20 antibody, rhuMAb CD20. Acetate (pH 5) formu
pH on the conformation of rhuMAb CD18 was investigated lations stored at 40 C. for one month demonstrated greater
using fluorescence spectroscopy (FIG. 17). The protein stability than those samples formulated in histidine (pH 5 or
appeared to lose tertiary Structure below pH 3, but is 35 6). The histidine formulations after accelerated temperature
unchanged in the range of pH 3 to 8. Storage became very opalescent and yellow in color. A
Based on the above observation, the effect of pH and buffering capacity of 10–30 mM acetate was sufficient to
protein concentration on the stability of rhuMAb CD18 was maintain the pH at 5.0. The effective amount of tonicity
then investigated using a central composite design protocol. modifier needed to Stabilize the antibody against freeze or
Formulations containing 10 mM Na citrate, 8% trehalose, 40 thermal induced aggregation was compared using Sodium
0.05% TWEEN 20TM containing one of the following con chloride (NaCl) or trehalose. Trehalose was found to protect
ditions were prepared: 0.5 mg/mL (pH 4.5), 5 mg/mL (pH3, the formulation from freeze induced aggregation, particu
4.5, 6), 10 mg/mL (pH 4, 5), 25 mg/mL (pH 4.5). Samples larly at levels >134 mM (500:1 molar ratio). The trehalose
were placed at 40 C. and -70° C., and analyzed after 2 formulations (67-270 mM) were much more effective than
month by IEX (FIG. 18A) and SEC (FIG. 18B). Again, pH 45 NaCl in stabilizing formulations placed at 40 C. as evi
5 was found to be the most stable condition for the protein, denced by the clarity of the solution. These results led to the
irrespective of protein concentration. development of a Stable prototype liquid multidose formu
Long Term Stability of rhuMAb CD18 Formulation: To lation comprising 40 mg/mL rhuMAb CD20, 25 mM
address the long term stability of the F2 formulation, dupli acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02%
cate samples in 10 mM Na acetate, 8% trehalose, 0.01% 50 polysorbate 20 at pH 5.0 that has a minimum shelf life of
TWEEN 20TM, pH 5 were prepared in 3 cc glass vials and two years storage at 2-8 C.
placed on Stability at the indicated temperatures. Analysis by MATERIALS AND METHODS
SEC (FIG. 19A) and MAC-1 binding (FIG. 19C) indicates
no change in the size distribution, aggregation State, or RhuMAb CD20: The bulk material used in all studies was
bioactivity of the protein for up to 43 weeks at 5°C; stability 55 composed of 27 mg/mL rhuMAb CD20 in 50 mM Tris and
up to ~1 mo at 30° C. is indicated. IEX shows formation of 100 mM sodium chloride at pH 7.5 or 2.7 mg/mL rhuMAb
acidic peaks (~1% after 43 weeks) that were identified to be CD20 in 25 mM Tris and 50 mM sodium chloride at pH 6.0.
deamidation by peptide mapping/MS (FIG. 19B). The bulk was stored aseptically at 2-8 C. in the absence of
light.
CONCLUSIONS 60 Buffer exchange: The exchange of the bulk rhuMAb
Preliminary data Suggested that the primary reaction in CD20 into different test buffers was done by dialysis at 2-8
the purified rhuMAb CD18 was cleavage to species approxi C. using Spectra/Por(R 7 membranes (MWCO=8 Kda)
mately half the MW of the starting material, and that this which were rinsed thoroughly with deionized water before
reaction, as well as generation of acidic peaks on IEX and use. One Volume of antibody was dialyzed against a mini
earlier eluting peaks on HIC and RP-HPLC, were all mini 65 mum of 10 volumes of the appropriate test buffer. This
mized at pH 5 (compared to pH 6), and in trehalose process was repeated three to four times within a one day
(compared to salt or mannitol). The smaller MW species period. To obtain the final concentration of 40 mg/mL, the
US 6, 171586 B1
41 42
antibody was further concentrated using an Amicon UF/DF HIC HPLC: Samples were diluted to 10 mg/mL with
cell containing a YM30 membrane (MWCO=30 KDa). Due formulation buffer before being assayed. The antibody was
to the lower Starting concentration of the 2.7 mg/mL digested with carboxypeptidase and papain prior to analysis
material, this material was first concentrated to ~40 mg/mL of the final fragments. The method uses a TSK-GEL butyl
and then dialyzed against the appropriate test buffer. After NPR (4.6x35 mm) column. The temperature of the column
the target concentration was reached and the buffer eXchange is controlled at 35 C. during the assay. Elution of the
completed, trehalose, benzyl alcohol and polySorbate 20 antibody fragments is induced by changes in the ammonium
were added to the final concentrations described in Tables 15
to 17 below. The liquid multdose C2B8 candidate formula Sulfate gradient. The total run time of the assay is forty
tion was 40 mg/mL rhuMAb CD20 formulated in 25 mM minutes and the flow rate is 1 ml/min. A protein load of 5-10
acetate, 150 mM trehalose, 0.9% benzyl alcohol and 0.02% 1O tug is injected and the detection is monitored by UV absor
polysorbate 20 at pH 5.0. The formulation was then sterile bance at 214 nm.
filtered through a 0.2u membrane and the concentration was UV spectrophotometric Scan: For protein concentration
determined by UV spectrophotometric scan. A 0.5 ml of determination, samples were accurately diluted 1:100 with
each final formulation was then filled into Sterile 3 cc glass formulation buffer. The absorbance at 280,320 and 350 were
Vials, Stoppered with teflon faced grey butyl rubber Stoppers 15 read with an Hewlett Packard 8451A diode array spectro
and then capped with crimp Seals. photometer against the same formulation buffer as blank.
The protein concentration was calculated by Subtracting
TABLE 1.5 A from Aso and dividing by an extinction coefficient of
1.7.
pH and buffer species comparison study
For turbidity evaluation, Samples were Scanned without
Concentration of Concentration
rhuMAb CD20 of buffer Storage Temperature
dilution on an Hewlett Packard 8451A diode array spectro
(mg/mL) species (mM) pH ( C.) photometer and the average absorbance in the range of
340-360 nm was determined. Water was used as the blank.
40 50 mM Acetate 5.0 2-8, 40, 50 Accelerated stability studies: The vials were stored
40 50 mM Histidine 5.0 2-8, 40, 50 25
40 50 mM Histidine 6.O 2-8, 40, 50 upright in temperature-controlled rooms or incubators at
2–8, 30, 40 or 50° C. Two to three vials were removed at
All formulations contain 150 mM trehalose, 0.9% benzyl alcohol and finite times and the protein degradation monitored by the
0.02% polysorbate 20. stability indicating assays (SEC HPLC, HIC HPLC, UV
Spectrophotometric Scan to determine protein concentration
TABLE 16 and turbidity, as well as pH measurement). In addition,
Samples were Subjected to the complement dependent cell
Acetate buffering capacity study cytotoxicity assay as described below to assess bioactivity.
Concentration of rhuMAb Freeze-thaw Studies: Samples were exposed to a mini
CD20 (mg/mL) mM Acetate Storage temperature (C.) 35 mum of two hours of freezing at -70° C. followed by a room
temperature thaw (<45 minutes). Each cycle was composed
40 1O 2-8, 40 of one freeze followed by one thaw excursion. Three vials
40 15 2-8, 40
40 2O 2-8, 40 were removed per formulation after one, three and five
40 25 2-8, 40 consecutive freeze-thaw cycles and the Stability monitored
40 3O 2-8, 40 40 by SEC HPLC and UV spectrophotometric scan to measure
All formulations contain 150 mM trehalose, 0.9% benzyl alcohol and
the turbidity and protein concentration.
0.02% polysorbate 20 at pH 5.0. Complemnent Dependent Cytotoxicity Assay (CDC): The
bioactivity of the stability samples was determined by the
CDC assay described in Gazzano Santoro et al., J. Immunol.
TABLE 1.7 45 Meth. 202:163–171 (1997), except that human complement
Effective ratio of tonicity modifier: rhuMAb CD20 study (rather than rabbit complement) was used. The percent
bioactivity of the test Sample was determined as follows,
Molar ratio of mM trehalose or sodium
Tonicity modifier sugarfsalt:rhuMAb CD20 chloride (CDC assay mg/mL of samplef
50
% bioactivity = protein concentration of sample) X 100
Trehalose O O
Trehalose 250:1 67 (CDC assayy mg
mg/mL of referencef
Trehalose SOO:1 134
Trehalose 1OOO:1 267 protein concentration reference)
Sodium Chloride SOO:1 134
Sodium Chloride 1OOO:1 267
55 The protein concentration of the test Sample and reference
A). All formulations contained 40 mg/mL rhuMAb CD20, 20 mM acetate, control were determined by UV spectrophotometric Scan.
0.9% benzyl alcohol and 0.02% polysorbate 20 at pH 5.0.
B). A freeze-thaw comparison study was completed for the trehalose for RESULTS AND DISCUSSION
mulations. The stability of all formulations at 2-8 and 40° C. was con
ducted in parallel. pH and buffer species: Decreasing the pH from 7.5 to 5.0
SEC HPLC: Samples were diluted to 10 mg/mL with 60 of 40 mg/mL rhuMAb CD20 formulated in histidine and
formulation buffer before being assayed. The method uses a trehalose virtually eliminated oxidation of the antibody even
TSKG3000 SWXL column (TosoHaas) with a mobile phase after two months storage at 40° C. (FIG.24B). There is also
consisting of 0.2M potassium phosphate, 0.25M potassium a decline in the rate of aggregation but the difference below
chloride, pH.7. The Socratic flow rate is 0.5 mLlmin with a pH 6.5 is slight (FIG.24A). Further reduction in aggregation
total run time of 30 minutes. The amount of protein injected 65 rate may require a decrease in the protein concentration. A
is 200 up and the UV absorbance at 280 nm is used as the Stable formulation appears to require a pH in the acidic
mode of detection. range.
US 6, 171586 B1
43 44
To differentate between the effect of buffer species and the
effect of pH, histidine (5 or 6) and acetate (5), multdose TABLE 1.9
formulations were compared after storage at 2–8, 40 or The effect of buffer species and pH on the percentage
50° C. The samples were stored at 50° C. as a way of quickly unoxidized Fe and percentage monomer of 40 mg/mL rhuMAb
determining the relative stability between the formulations CD20 multidose formulations containing 50 mM acetate or
with the caveat that the degradation observed was not histidine, 150 mM trehalose, 0.9% benzyl alcohol and 0.02%
polysorbate 20 at pH S or 6.
necessarily predictive of that seen at 2-8 C. Storage.
%. Unoxidized Fc % Monomer
Formulation Temperature (HIC HPLC) (SEC HPLC)
T=O
The visual clarity of the formulations and the turbidity as
measured by UV spectrophotometric scan (340–360 nm) Acetate pH 5 2-8 C. 93.9 99.4
after four (50° C.) and eight (2–8, 40°C.) weeks storage is Histidine pH 5 2-8 C. 99.1 99.4
Histidine pH6 2-8 C. 99.3 98.5
described in Tab Both histidine formulations were more 15 T = 8 weeks
opalescent than the acetate at all temperatures Studied. After
Acetate pH 5 40° C. 96.5 90.4
two weeks storage at 50 C., the histcline at pH 5 had formed Histidine pH 5 40° C. 87.O 87.4
a Solid opaque gel while the Sibling formulation at pH 6 was Histidine pH 6 40° C. 81.7 93.9
visually cloudy and yellow in color by four weeks. The
histidine formulations stored at 40 C. also turned yellow. Since the acetate pH 5 formulation had only a slightly
Without being bound to any one theory, the color formation higher aggregation rate, did not turn yellow upon Storage at
is likely due to the oxidation of histidine and is more high temperatures and had the greatest clarity under all
apparent in this study due to the high concentration of conditions Studied, it was chosen as the buffer Species and
histidine used (50 mM). No differences were observed at 25
pH of choice for all Subsequent liquid C2B8 multidose
2-8 C. storage relative to the initial timepoint. formulation Screens.
Amount of buffering Species: The amount of acetate
TABLE 1.8 which maintained the pH of a 40 mg/mL C2B8 multidose
formulation at 5.0 was determined. Summarized in Table 20
The effect of buffer species and pH on the appearance is the effect on pH as the acetate buffer concentration was
and clarity of 40 mg/mL rhuMAb CD20 multidose formulations increased from 10 to 30 mM. At two and four weeks there
containing 50 mM acetate or histidine, 150 mM trehalose,
0.9% benzyl alcohol and 0.02% polysorbate 20 at pH S or 6. appeared to be a slight advantage in Staying above 15 mM
acetate, although this did not hold true upon long term
Turbidity Storage. No change was seen in the pH in any of the test
Avg. O.D. formulations studied after one years storage at 2-8 C. A
Formulation Temperature Appearance 340-360 nm. 35
range of 10 to 30 mM acetate is sufficient to maintain the pH
T = 8 weeks at 5.0.
Acetate pH 5 ea 0.051 - 0.001
TABLE 2.0
Histidine pH 5 ea 0.065 + 0.0002
Histidine pH 6 ea O.O68 O.OO1
40
Acetate pH 5 palescent O.16 O.OO2 The effect of acetate buffer concentration on maintaining
Histidine pH 5 Light yellow, 0.28 0.015 the pH of liquid rhuMAb CD20 multidose formulations at 5.0.
Opalescent The formulations were composed of 40 mg/mL rhuMAb CD20, 10
Histidine pH 6 Light yellow, O.36 O.O26 to 30 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol,
Opalescent 0.02% polySOrbate 20 at pH 5.0.
T
o
= 4 weeks 45
mM pH (weeks
Acetate pH 5 50° C. Very Opalescent O.61 O.OO7
Histidine pH 5 50° C. Firm opaque gel ND1 Acetate O 2 4 56
Histidine pH 6 50° C. Cloudy, yellow solution 1.30 - 0.2O
2-8 C.
"The turbidity was not determined (N/D) due to the gelation of the 1O S.O3 5.12 5.11 S.OO
sample. 50
15 5.07 5.12 5.14 5.06
2O 5.04 5.04 5.04 S.OO
25 S.O1 S.OO 5.02 4.98
3O S.O1 S.OO 5.02 4.98
40° C.
The stability was also monitored by HIC and SEC HPLC
55
methods. After eight weeks storage at 40 C., the acetate pH 1O 5.12 5.14
15 5.10 5.17
5 formulations were unchanged while the histidine pH 6 2O 5.08 5.10
formulations had a 18 percent reduction in unoxidized Fc 25 5.05 5.05
relative to the initial bmepoint (Table 19). The percentage 3O S.O3 5.05
monomer decreased in all formulations stored at 40 C. with
the histidine pH 6 being slightly more stable. This reduction Ratio of tonicity modifier: protein: The addition of tre
was attributed to the formation of a high molecular weight halose was beneficial to the stability of rhuMAb CD20
aggregate(s) eluting at the Void Volume, a lagging shoulder multdose formulations after multiple freeze/thaw cycles
on the monomer peak and lower molecular weight Species. (FIG.25). A decrease in the formation of Soluble aggregates
The protein concentration and pH were also measured, 65 was observed as the concentration of trehalose in the for
except in the case of gelation, and no changes were observed mulation was increased. At a ratio of 500 moles trehalose:1
over the duration of the study. mole C2B8 (134 mM trehalose), the percentage of aggregate
US 6, 171586 B1
45 46
formed was only 0.5% after five freeze-thaw cycles while Stability in the liquid State. The candidate liquid multidose
the control formulation containing no isotonicity modifier formulation preferably contains at least 500:1 molar ratio of
formed 4% aggregate. Having at least a 500 fold molar trehalose to C2B8.
excess of trehalose in the liquid C2B8 formulation offered
Sufficient protection against freeze induced aggregation. Stability of candidate liquid multidose formulation: Based
The ability of trehalose to stabilize rhuMAb CD20 during on the aforementioned Studies, a protoype liquid multidose
accelerated temperature Storage was Studied and compared formulation composed of 40 mg/mL rhuMAb CD20, 25 mM
to Sodium chloride. acetate, 150 mM trehalose, 0.9% benzyl alcohol and 0.02%
FIG. 26 describes the effect of excess molar ratios of polysorbate 20 at pH 5.0 was placed on stability at 2-8, 30
trehalose or sodium chloride on the clarity of test formula C. and 40° C. The stability profile by SEC HPLC at each
tions stored at 40 C. Sodium chloride is deleterious to the temperature studied is shown in FIG. 28. Although the rate
stability. After two weeks storage at 40 C., the 500:1 ratio of aggregation is slightly faster at 40 C. for the multidose
sample had an O.D. of 0.44. At a 1000:1 molar ratio, the formulation (40 mg/mL) compared to the reference control
Sodium chloride containing formulation Separated into a two (10 mg/mL rhuMAb CD20, 25 mM citrate, 150 mM sodium
phase System composed of an opaque gel covered with an 15 chloride, 0.07% polysorbate 80 at pH 6.5), no decrease in
opalescent fluid on top. In contrast, there was little change percentage monomer was observed upon Storage at 2-8 C.
in the solution clarity of the samples containing 0 to 1000:1 for two years. The bioactivity of the two year old 2-8 C.
molar ratio of trehalose even after one months storage at 40 samples was 99.2% relative to the reference control as
C. Although the 2-8 C. formulations were unchanged from determined by the CDC assay.
the initial bmepoint, the Sodium chloride containing Samples
were more opalescent. CONCLUSIONS
The effect of trehalose on minimizing the Soluble aggre
gate formation was assessed by SEC HPLC (FIG. 27). No The above Screening Studies indicated that a stable high
differences were observed between the trehalose formula 25 concentration rhuMAb CD20 liquid multidose formulation
tions and the negative control which contained no tonicity was possible by buffering with acetate, maintaining the pH
modifier. The formulations appeared to degrade at the same at 5 and including preferably at least about 500 moles of
rate to the same products upon Storage at 40 C. The pH and trehalose per mole of antibody. The preferred liquid multi
concentration were also maintained over the duration of the dose configuration is composed of 40 mg/mL rhuMAb
Study . CD20, 25 mM acetate, 150 mM trehalose, 0.9% benzyl
The presence of trehalose is beneficial in minimizing alcohol and 0.02% polysorbate 20 at pH 5 and has a shelf life
freeze-induced aggregation and is not deleterious to the of two years at 2-8 C.
SEQUENCE LISTING
(1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES: 11
(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 241 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi). SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
Glu Wall Glin Leu Wall Glu Ser Gly Gly Gly Telu Wall Glin Pro Gly
1 5 10 15
Gly Ser Leu Arg Teu Ser Cys Ala Thir Ser Gly Tyr Thr Phe Thr
20 25 30
Glu Thir Met His Trp Met Arg Glin Ala Pro Gly Gly Teu
35 40 45
Glu Trp Wall Ala Gly Ile Asn. Pro Asn Gly Gly Thr Ser His
5O 55 60
Asn Glin Arg Phe Met Asp Arg Phe Thir Ile Ser Wall Asp Ser
65 70 75
Thr Ser Thir Ala Tyr Met Glin Met Asn Ser Lieu Ala Glu Asp
85 90
Thr Ala Val Tyr Tyr Ala Arg Trp Arg Gly Telu Asn Gly
95 100 105
US 6, 171586 B1
47 48
-continued
Phe Asp Val Arg Tyr Phe Asp Val Trp Gly Glin Gly Thr Leu Val
10 15 20
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
25 30 35
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thir Ser Gly Val His Thr Phe Pro Ala Val
Leu Glin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu Gly. Thr Glin Thr Tyr Ile Cys Asn Val Asn
200 2O5 210
His Lys Pro Ser Asn. Thir Lys Val Asp Llys Lys Val Glu Pro Lys
215 220 225
Ser Cys Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
230 235 240
Teu
241
(2) INFORMATION FOR SEQ ID NO: 2:

(xi). SEQUENCE DESCRIPTION: SEQ ID NO:2:
Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glin Asp Ile Asn
2O 25 30
Asn Tyr Lieu. Asn Trp Tyr Glin Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Leu Leu Ile Tyr Tyr Thr Ser Thr Leu. His Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thir Ile
65 70 75
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin
8O 85 9O
Gly Asn Thr Leu Pro Pro Thr Phe Gly Glin Gly Thr Lys Val Glu
95 OO O5
Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
10 15 20
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Lieu
25 30 35
Lieu. Asn. Asn. Phe Tyr Pro Arg Glu Ala Lys Val Glin Trp Llys Val
40 45 5O
Asp Asn Ala Leu Glin Ser Gly Asn. Ser Glin Glu Ser Val Thr Glu
55 60 65
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
70 75 8O
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
85 90 95
US 6, 171586 B1
49 SO
-continued
Val Thr His Glin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
200 2O5 210
Arg Gly Glu Cys

214
(2) INFORMATION FOR SEQ ID NO:3:

Leu Gly Gly Arg Met Lys Glin Leu Glu Asp Llys Val Glu Glu Lieu
1 5 10 15
Leu Ser Lys Asn Tyr His Leu Glu Asn. Glu Val Ala Arg Lieu Lys
2O 25 30
Lys Lieu Val Gly Glu Arg

35 36

(xi). SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro
1 5 10 11

His Glin Ser Leu Gly Thr Glin
1 5 7

(xi). SEQUENCE DESCRIPTION: SEQ ID NO: 6:
His Glin Asn Lieu Ser Asp Gly Lys
1 5 8
(2) INFORMATION FOR SEQ ID NO: 7 :

(xi). SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
His Glin Asn. Ile Ser Asp Gly Lys
1 5 8

US 6, 171586 B1
S1 52
-continued
(i) SEQUENC E CHARACTERISTICS

(xi) SEQUENC E DESCRIPTION: SEQ ID NO :8:
Wall Ile Ser Ser His Lieu Gly Glin
1 5 8

(i) SEQUENC E CHARACTERISTICS
(A) LENGTH: 2143 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(xi) SEQUENC E DESCRIPTION: SEQ ID NO: 9:
GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC 5O
TCATTGCTGA GTTGTTATTT AAGCTTTGGA GATTATCGTC ACTGCAATGC 100
TTCGCAATAT GGCGCAAAAT GACCAACAGC GGTTGATTGA TCAGGTAGAG 15 O
GGGGCGCTGT ACGAGGTAAA GCCCGATGCC AGCATTCCTG ACGACGATAC 200
GGAGCTGCTG CGCGATTACG TAAAGAAGTT ATTGAAGCAT CCTCGTCAGT 25 O
AAAAAGTTAA TCTTTTCAAC AGCTGTCATA AAGTTGTCAC GGCCGAGACT 3OO
TATAGTCGCT TTGTTTTTAT TTTTTAATGT ATTTGTAACT AGAATTCGAG 350
CTCGCCGGGG ATCCTCTAGA GGTTGAGGTG ATTTTATGAA AAAGAATATC 400
GCATTTCTTC TTGCATCTAT GTTCGTTTTT TCTATTGCTA CAAACGCGTA 450
CGCTGATATC CAGATGACCC AGTCCCCGAG CTCCCTGTCC GCCTCTGTGG 5 OO
GCGATAGGGT CACCATCACC TGTCGTGCCA GTCAGGACAT CAACAATTAT 550
CTGAACTGGT ATCAACAGAA ACCAGGAAAA GCTCCGAAAC TACTGATTTA 600
CTATACCTCC ACCCTCCACT CTGGAGTCCC TTCTCGCTTC TCTGGTTCTG 650
GTTCTGGGAC GGATTACACT CTGACCATCA GCAGTCTGCA ACCGGAGGAC FOO
TTCGCAACTT ATTACTGTCA GCAAGGTAAT ACTCTGCCGC CGACGTTCGG 750
ACAGGGCACG AAGGTGGAGA TCAAACGAAC CCATCTGTCT 800
TCATCTTCCC GCCATCTGAT GAGCAGTTGA AATCTGGAAC TGCCTCTGTT 85 O
TGAATAACTT CTATCCCAGA GAGGCCAAAG TACAGTGGAA 9 OO
GGTGGATAAC GCCCTCCAAT CGGGTAACTC CCAGGAGAGT GTCACAGAGC 95 O
AGGACAGCAA GGACAGCACC TACAGCCTCA GCAGCACCCT GACGCTGAGC OOO
AAAGCAGACT ACGAGAAACA CAAAGTCTAC GCCTGCGAAG TCACCCATCA
GGGCCTGAGC TCGCCCGTCA CAAAGAGCTT CAACAGGGGA GAGTGTTAAG 100
CTGATCCTCT ACGCCGGACG CATCGTGGCG CTAGTACGCA AGTTCACGTA 15 O
AAAACGGTAT CTAGAGGTTG AGGTGATTTT ATGAAAAAGA ATATCGCATT 200
TCTTCTTGCA TCTATGTTCG TTTTTTCTAT TGCTACAAAC GCGTACGCTG 25 O
AGGTTCAGCT GGTGGAGTCT TGGTGCAGCC AGGGGGCTCA 3OO
CTCCGTTTGT CCTGTGCAAC TTCTGGCTAC ACCTTTACCG AATACACTAT 350
GCACTGGATG CGTCAGGCCC CGGGTAAGGG CCTGGAATGG GTTGCAGGGA 400
TTAATCCTAA AAACGGTGGT ACCAGCCACA ACCAGAGGTT CATGGACCGT 450

US 6, 171586 B1
S3
-continued
TTCACTATAA. GCGTAGATAA, ATCCACCAGT ACAGCCTACA. TGCAAATGAA 5 OO
CAGCCTGCGT, GCTGAGGACA CTGCCGTCTA TTATTGTGCT AGATGGCGAG 550
GCCTGAACTA CGGCTTTGAC GTCCGTTATT TTGACGTCTG GGGTCAAGGA 600
ACCCTGGTCA, CCGTCTCCTC. GGCCTCCACC AAGGGCCCAT, CGGTCTTCCC 650
CCTGGCACCC. TCCTCCAAGA, GCACCTCTGG GGGCACAGCG GCCCTGGGCT FOO
GCCTGGTCAA. GGACTACTTC CCCGAACCGG, TGACGGTGTC. GTGGAACTCA 750
GGCGCCCTGA CCAGCGGCGT, GCACACCTTC CCGGCTGTCC TACAGTCCTC. 800
AGGACTCTAC. TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC AGCAGCTTGG 85 O
GCACCCAGAC CTACATCTGC AACGTGAATC. ACAAGCCCAG CAACACCAAG 9 OO
GTCGACAAGA AAGTTGAGCC CAAATCTTGT, GACAAAACTC. ACACATGCCC 95 O
GCCGTGCCCA GCACCAGAAC TGCTGGGCGG CCGCATGAAA CAGCTAGAGG 2OOO
ACAAGGTCGA, AGAGCTACTC. TCCAAGAACT ACCACCTAGA GAATGAAGTG 2O5 O
GCAAGACTCA AAAAGCTTGT, CGGGGAGCGC TAAGCATGCG ACGGCCCTAG 2100
AGTCCCTAAC GCTCGGTTGC CGCCGGGCGT TTTTTATTGT TAA 21 4 3
(2) INFORMATION FOR SEQ ID NO: 10 :

(xi). SEQUENCE DESCRIPTION: SEQ ID NO : 10
Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe
-23 -20 -15 -10
Ser Ile Ala Thr Asn Ala Tyr Ala Asp Ile Gln Met Thr Glin Ser
-5 1 5
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
10 15 20
Cys Arg Ala Ser Glin Asp Ile Asn. Asn Tyr Lieu. Asn Trp Tyr Glin
25 30 35
Glin Lys Pro Gly Lys Ala Pro Llys Lieu Lieu. Ile Tyr Tyr Thr Ser
40 45 5O
Thr Leu. His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
55 60 65
Gly Thr Asp Tyr Thr Leu Thir Ile Ser Ser Leu Gln Pro Glu Asp
70 75 8O
Phe Ala Thr Tyr Tyr Cys Glin Glin Gly Asn Thr Leu Pro Pro Thr
85 9O 95
Phe Gly Glin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
OO 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
15 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
30 135 140
Glu Ala Lys Val Glin Trp Llys Val Asp Asn Ala Leu Glin Ser Gly
45 15 O 155
Asn Ser Glin Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser Thr
60 1.65 170
Tyr Ser Lieu Ser Ser Thr Lieu. Thir Lieu Ser Lys Ala Asp Tyr Glu
US 6, 171586 B1
SS
-continued
175 18O 185
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Glin Gly Lieu Ser
190 195 200
Ser Pro Val Thr Lys Ser Phe Asin Arg Gly Glu Cys
210 214

(xi). SEQUENCE DESCRIPTION: SEQ ID NO : 11:
Met Lys Lys Asn Ile Ala Phe Leu Lieu Ala Ser Met Phe Wall Phe
-23 -20 -15 -10
Ser Ile Ala Thr Asn Ala Tyr Ala Glu Wall Glin Teu Wall Glu Ser
-5 1
Gly Gly Gly Telu Wall Glin Pro Gly Gly Ser Teu Arg Teu Ser Cys
10 15 20
Ala Thr Ser Gly Thr Phe Thr Glu Thr Met His Trp Met
25 30 35
Arg Glin Ala Pro Gly Gly Telu Glu Trp Wall Ala Gly Ile Asn
40 45 5O
Pro Asn Gly Gly Thr Ser His Asn Glin Arg Phe Met Asp
55 60 65
Phe Thr Ile Ser Wall Asp Ser Thr Ser Thr Ala Met Glin
70 75 8O
Met Asn Ser Telu Arg Ala Asp Thr Ala Wall Ala
85 9O 95
Arg Trp Arg Gly Teu Asn Gly Phe Asp Wall Arg Phe
OO O5 10
Wall Trp Glin Gly Thr Teu Thr Wall Ser Ser Ser Thr
Gly Ser Wall Phe Pro Ala Pro Ser Ser Ser Thr
Ser Gly Thr Ala Ala Teu Telu Wall Phe
Pro Glu Wall Thr Wall Ser Asn Ser Gly Ala Thr Ser
Gly Wall Thr Phe Pro Ala Telu Glin Ser Ser Teu Tyr
Ser Telu Ser Ser Wall Wall Thr Pro Ser Ser Ser Teu Thr
9O 200
Glin Thr Ile Asn Wall Asn His Pro Ser Asn Thr Lys
2O5 210 215
Wall Asp Lys Wall Glu Pro Lys Ser Asp Thr His Thr
220 225 230
Pro Pro Pro Ala Pro Glu Telu Telu Gly Arg Met Lys
235 240 245
Glin Telu Glu Asp Wall Glu Glu Telu Telu Ser Asn His
25 O 255 260
Teu Glu Asn Glu Wall Ala Arg Telu Teu Wall Gly Glu Arg
265 27 O 275 277
US 6, 171586 B1
57 58
What is claimed is: 19. The formulation of claim 18 wherein the preservative
1. A stable aqueous pharmaceutical formulation compris is benzyl alcohol.
ing a therapeutically effective amount of an antibody not 20. The formulation of claim 15 wherein the antibody is
Subjected to prior lyophilization, an acetate buffer from present in an amount of about 30-50 mg/mL.
about pH 4.8 to about 5.5, a surfactant and a polyol, wherein 21. The formulation of claim 20 wherein the buffer is
the formulation lacks a tonicifying amount of Sodium chlo 10–30 mM sodium acetate at pH 5, the polyol is trehalose in
ride. an amount of about 2-10% w/v, the surfactant is polysorbate
2. The formulation of claim 1 which is isotonic. in an amount of about 0.01-0.1% V/V, wherein the formu
3. The formulation of claim 1 which is stable at a lation further comprises benzyl alcohol as a preservative and
temperature of about 2-8 C. for at least one year. wherein the formulation is stable at a temperature of about
4. The formulation of claim 1 which is stable following 2-8 C. for at least two years.
freezing and thawing of the formulation. 22. An article of manufacture comprising a container
5. The formulation of claim 1 which is stable at about 30 holding a Stable aqueous pharmaceutical formulation com
C. for at least one month. prising a therapeutically effective amount of an antibody not
6. The formulation of claim 1 wherein the polyol is a 15
Subjected to prior lyophilization, an acetate buffer from
nonreducing Sugar. about pH 4.8 to about 5.5, a surfactant and a polyol, wherein
7. The formulation of claim 6 wherein the nonreducing the formulation lacks a tonicifying amount of Sodium chlo
ride.
Sugar is trehalose. 23. A method for Stabilizing an antibody in an aqueous
8. The formulation of claim 6 wherein the nonreducing pharmaceutical formulation by combining a therapeutically
Sugar is Sucrose. effective amount of an antibody not Subjected to prior
9. The formulation of claim 1 wherein the antibody is an lyophilization, an acetate buffer from about pH 4.8 to about
antibody fragment.
10. The formulation of claim 9 wherein the antibody 5.5, a Surfactant and a polyol, wherein the formulation lackS
fragment is a F(ab'). a tonicifying amount of Sodium chloride.
11. The formulation of claim 1 wherein the antibody binds 25
24. A Stable aqueous pharmaceutical formulation com
CD18. prising a therapeutically effective amount of an antibody not
12. The formulation of claim 1 which is stable at a subjected to prior lyophilization, a 10-30 mM acetate buffer
temperature of about 2-8 C. for at least two years. at pH 5.0, a Surfactant, and trehalose at a concentration of at
13. The formulation of claim 1 wherein the antibody least 134 mM, wherein the formulation lacks a tonicifying
amount of Sodium chloride.
concentration in the formulation is from about 0.1 to about
50 mg/mL. 25. The formulation of claim 24 wherein the antibody
binds CD18 or CD2O.
14. The formulation of claim 1 wherein the Surfactant is
26. The formulation of claim 25 wherein the CD18
a polySorbate. binding antibody is a F(ab')2 fragment.
15. The formulation of claim 1 wherein the antibody binds 27. An article of manufacture comprising a container
CD2O. 35
16. The formulation of claim 15 wherein the acetate is holding the formulation of claim 25.
present in an amount of about 5–30 mM. 28. The formulation of claim 1, wherein the acetate buffer
17. The formulation of claim 16 wherein the acetate is is at pH 5.0.
present in an amount of 10–30 mM. 29. The formulation of claim 28, wherein the antibody
binds CD20.
18. The formulation of claim 15 further comprising a 40
preservative.

United States Patent: (10) Patent No.: US 6,171,586 B1

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USOO6171586B1

(12) United States Patent (10) Patent No.: US 6,171,586 B1

(54) ANTIBODY FORMULATION WO 94/26302 11/1994 (WO).

OTHER PUBLICATIONS Mileski et al., “Inhibition of CD18-dependent neutrophil

Hydrophilic & Flexible

O 50 100 150 200 FI G 2 A

CDR CDR CDR

Hydrophilic & Flexible

Storage Temperature (C)

Retention Time (Minutes)

Storage Temperature (C)

Storage Time (Weeks)

0.0031 0.0032 0.0034 0.0035 0.0036 00037

3 4.1 4.1 4. 5 4.5 4.5 5 5 6

l GAACAACT TCTCCATACT TTGGATAAGG AAATACAGAC AGAAAAATC TCATTGCTGA

W3110 K-12F lambda NrrinD-rrTEl

16C9 W3110 Afhua AphoA A(argF-lac) deoC

33B6 W3110 AthuA AphoA A(argF-lac) deoC AdegPilvG

PRIMARY INOCULUM, 14-18 hours

PRODUCTION VESSEL, temperature and pH controlled

250 T. 1 500 T: 1 1 OOOT: 1

100 200 300 400 50 O 600 700

are predicted to be most reactive, reactivity at ASn 84 is most 4

*Extinction coefficient was later changed to 1.45 mL/mg/cm.

These protein Solutions were then diluted, using reserved

Dialysis 10% Dialy. Starting Final *Final

* Measured after filtration and filling.

The formulations were sterile filtered using 150 mL TABLE 6B

UV SpectroScopy: Spectroscopic analysis of the Samples

-70 O.976 O.O98 O.906 O.112 0.952 O.918 O.433

(1) GENERAL INFORMATION:

(2) INFORMATION FOR SEQ ID NO : 1 :

(2) INFORMATION FOR SEQ ID NO: 2:

Arg Gly Glu Cys

(2) INFORMATION FOR SEQ ID NO:3:

Lys Lieu Val Gly Glu Arg

(2) INFORMATION FOR SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7 :

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENC E CHARACTERISTICS

(2) INFORMATION FOR SEQ ID NO: 9:

TCATTGCTGA GTTGTTATTT AAGCTTTGGA GATTATCGTC ACTGCAATGC 100

TTCGCAATAT GGCGCAAAAT GACCAACAGC GGTTGATTGA TCAGGTAGAG 15 O

GGGGCGCTGT ACGAGGTAAA GCCCGATGCC AGCATTCCTG ACGACGATAC 200

GGAGCTGCTG CGCGATTACG TAAAGAAGTT ATTGAAGCAT CCTCGTCAGT 25 O

AAAAAGTTAA TCTTTTCAAC AGCTGTCATA AAGTTGTCAC GGCCGAGACT 3OO

TATAGTCGCT TTGTTTTTAT TTTTTAATGT ATTTGTAACT AGAATTCGAG 350

CTCGCCGGGG ATCCTCTAGA GGTTGAGGTG ATTTTATGAA AAAGAATATC 400

GCATTTCTTC TTGCATCTAT GTTCGTTTTT TCTATTGCTA CAAACGCGTA 450

CGCTGATATC CAGATGACCC AGTCCCCGAG CTCCCTGTCC GCCTCTGTGG 5 OO

GCGATAGGGT CACCATCACC TGTCGTGCCA GTCAGGACAT CAACAATTAT 550

CTGAACTGGT ATCAACAGAA ACCAGGAAAA GCTCCGAAAC TACTGATTTA 600

CTATACCTCC ACCCTCCACT CTGGAGTCCC TTCTCGCTTC TCTGGTTCTG 650

GTTCTGGGAC GGATTACACT CTGACCATCA GCAGTCTGCA ACCGGAGGAC FOO

TTCGCAACTT ATTACTGTCA GCAAGGTAAT ACTCTGCCGC CGACGTTCGG 750

ACAGGGCACG AAGGTGGAGA TCAAACGAAC CCATCTGTCT 800