Enzyme and Microbial Technology 6465 (2014) 4451

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Enzyme and Microbial Technology

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Production of monoclonal antibodies for breast cancer by HB8696

hybridoma cells using novel perfusion system
Shweta Kamthan a , James Gomes b,1 , Pradip K. Roychoudhury a,
a
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
b
Kusuma School of Biological Sciences, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India

a r t i c l e i n f o a b s t r a c t

Article history: Perfusion culture using spinlters have been used for the production of health-care products using mam-
Received 7 March 2014 malian cells culture. However, available spinlters are either highly prone to clog and/or are disposable
Received in revised form 24 May 2014 and hence affects product formation. To address these problems, a novel non-woven Bombyx mori silk
Accepted 7 July 2014
screen based spinlter module for clog-free extended perfusion culture of hybridoma cells has been
Available online 14 July 2014
designed. The module is versatile in nature and reusable, after autoclaving and replacement of used
polymeric membrane. Its application for clog-free extended perfusion culture was demonstrated by
Keywords:
comparative perfusion experiments of HB8696 cells with stainless-steel spinlter. HB8696 cells pro-
Breast cancer
Monoclonal antibody production
duce monoclonal antibodies (MAbs) 520C9 active against breast cancer oncoprotein. Silk spinlter was
Silk spinlter found to be less prone to clog with cells and debris owing to its negatively charged hydrophobic screen
Stainless-steel spinlter compared to the positively charged hydrophilic stainless-steel spinlter. Therefore, it provides extended
Filter clogging cell growth phase and production phase of up to 56 h and 40 h respectively and 57.4% increase in MAb
Extended perfusion operation productivity compared to the stainless-steel spinlter. The effect of different perfusion rates on MAb
production was studied and an optimal MAb productivity of 1.6 g L1 day1 was achieved.
2014 Elsevier Inc. All rights reserved.

1. Introduction vibrations [7], etc. Considering the fact that neutral or negatively
charged hydrophobic polymeric lter screens are less prone to clog,
Spinlters have been primarily used for the perfusion culture of polymeric spinlters such as ethylene-tetrauoroethylene (ETFE),
hybridoma cells due to their selective cell retention efciency and polyamide and polytetrauoroethylene (PTFE) screen based spin-
low shear stress on cells [18]. But the primary drawbacks associ- lter [9,10], polyester screen based spinlter P [16], etc. were
ated with existing spinlter systems are either high lter clogging also designed. However, most of these spinlters are disposable
and/or disposability. For instance, positively charged hydrophilic in nature.
stainless-steel spinlters are highly prone to clog with negatively Therefore, for improving the process productivity during per-
charged cells and cell debris [912] which lowers process pro- fusion culture of hybridoma cells, a novel perfusion system has been
ductivity. To circumvent this problem, many strategies have been designed. The biocompatibility of silk to mammalian cells [17] has
previously reported, such as the addition of deoxyribonuclease I to been used as a selective advantage in designing a novel non-woven
the culture medium [11], variation of perfusion rate, screen pore Bombyx mori silk membrane based reusable spinlter module for
size and rotational speed of the spinlter [13,14], application of clog-free extended perfusion operation of hybridoma cells. Since
neutral and hydrophobic lter screens [8,9,15] and use of ultrasonic breast cancer is one of the most widespread invasive cancers world-
wide and accounts for approximately 29% of all cancers in women
[18], hybridoma cells produces MAb against breast cancer were
used in this study as a model system. Comparative perfusion exper-
Abbreviations: MAb, monoclonal antibody; SS 316, stainless steel 316; DMEM,
Dulbeccos modied Eagles medium; FBS, foetal bovine serum; LDH, lactate dehy-
iments of HB8696 hybridoma cells that produce MAb 520C9 active
drogenase; IgG1 , immunoglobulin G subclass 1. against breast cancer oncoprotein C-erbB2, were performed using
Corresponding author at: Block 1, Room No. I-138, Department of Biochemical stainless-steel spinlter to demonstrate the applicability of silk
Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, spinlter for clog-free extended perfusion operation. To enhance
New Delhi 110016, India. Tel.: +91 11 2659 1011; fax: +91 11 26582282. the MAb productivity, the perfusion rate for high MAb productivity
E-mail addresses: jgomes@bioschool.iitd.ac.in (J. Gomes), pkrc@dbeb.iitd.ac.in
(P.K. Roychoudhury).
by HB8696 hybridoma cells was also optimized while keeping the
1
Tel.: +91 11 26591013. spinlter rotation speed below the critical shear stress of cells. The

http://dx.doi.org/10.1016/j.enzmictec.2014.07.002
0141-0229/ 2014 Elsevier Inc. All rights reserved.
 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451 45

variation in perfusion rate was chosen as the primary parameter

for improving process productivity because increase in perfusion
rate reduces nutrient depletion and waste product accumulation
that maximizes cell growth and productivity [19,20]. Nevertheless,
due to cell wash out and high shear stress on cells, at high perfusion
rates [20], this parameter needs to be optimized.

2. Materials and methods

2.1. Cell line and cultivation medium

Two cell lines: the non-adherent mousemouse hybridoma cell line HB8696
(obtained from ATCC, LGC Promochem Ltd, Bangalore), which produces monoclonal
antibody immunoglobulin G subclass 1 (IgG1) active against breast cancer onco-
protein C-erbB2 and the adherent human brosarcoma kidney cell line HT1080,
obtained from NCCS, Pune) were used in this study. HB8696 cells were grown in
Hybricare medium (ATCC, 46-X) and HT1080 cells were grown in Dulbeccos mod-
ied Eagles medium (DMEM; Gibco, USA). Both the mediums were supplemented
with PenicillinStreptomycin solution (5%, v/v; 100 IU/mL and 0.1 mg/mL, respec-
tively; SigmaAldrich Corp., MO) and heat inactivated foetal bovine serum (FBS; 20%,
v/v; Biological Industries, Israel). Cells were thawed from frozen vials and grown
in 25 cm2 tissue culture asks (Corning, USA) in a humidied 5% CO2 incubator
controlled at 37 C.

2.2. Design of reusable spinlter module

The reusable spinlter module for perfusion culture of hybridoma cells was
constructed using stainless-steel (SS) 316 and Teon (Indian patent application
number 2509/DEL/2012). Its cylindrical polymeric membrane supporting compo-
nent was made up of Teon with ne holes drilled in it that permits continuous
media exchange during perfusion culture. It has two detachable silicone rubber
O-rings and a SS-316 clamp to hold the polymeric lter membrane in place. The
cylindrical component was attached to a SS-316 base, having an SS-316 Allen screw
attached to it, which helps in its positioning at desired depth on the motor shaft of
the bioreactor (Fig. 1a and b).
All the materials used in its construction were biocompatible to mammalian
cells, impervious to cell culture media constituents, chemical and corrosion-
resistant, and withstand high temperature up to 121 C. Thus the designed spinlter
module is autoclavable and can be easily cleaned. The designed spinlter module
is reusable, as after completion of one perfusion run the same module can be used
again after autoclaving and replacement of used polymeric membrane with the new
one. The designed spinlter module is versatile in operation, as any biocompatible
polymeric membrane, selected according to the type and size of cells cultured, can
be mounted over it as a lter screen. The module is presently designed for a 2-L
bioreactor system, but it is amenable to scale-up to a higher volume (the data will Fig. 1. (a) 3D image of silk spinlter generated by Autodesk 3ds max software.
be communicated shortly). (A) Porous teon membrane support, (B) shaft holding cone of stainless steel with
Allen screw, (C) metallic clamp for xing the membrane, (D) top and bottom view
2.3. Characterization of silk membrane as lter screen of spinlter module, (E) top and bottom view of spinlter module with silk. (b)
Dimensions of different components of silk spinlter. The components clockwise
For selecting a non-woven Bombyx mori silk membrane as lter screen for reten- from the top left panel are porous teon membrane support, stainless steel shaft
tion of hybridoma cells, the following surface properties (wettability, surface charge holding cone, Allen screw for xing the module and stainless steel clamp for holding
density and pore size) of three different non-woven Bombyx mori silks: Seri-DSS, the membrane. All the dimensions are in centimetres.
Seri-FSS and BF27PV (obtained from Sericare, Bangalore) and their effects on cell
adhesion and proliferation were evaluated:
2.3.4. Cell attachment supporting nature of silk membranes
The mammalian cell attachment supporting nature of each silk membrane
2.3.1. Wettability
(Seri-DSS, Seri-FSS and BF27PV taken separately) was tested by culturing adherent
Wettability of both surfaces of different silk membranes was evaluated by
HT1080 cells on them in 24-well plates (Corning, USA). Growth medium used was
measuring their mean water contact angle value using contact angle goniometer
DMEM supplemented with 20% heat inactivated FBS and 5% penicillinstreptomycin
(DSA100, Kruss GmbH, Germany). For each measurement, droplet of 3 L of Milli-Q
solution. For each of the three membranes, two 24-well plates were used. The two
water was placed on 3 cm 3 cm strip of each membrane and the contact angle value
other 24-well plates without membrane were used as control. Together, these eight
was measured. Three measurements were done for each side of the membrane and
24-well plates constituted a set for one experiment. Three sets of these experiments
the mean value was calculated
were performed. In each of the wells, 1 cm diameter circular piece of membrane was
taken. The seeding density of 1.4 105 cells/mL was used for each well and incubated
2.3.2. Surface charge density in a humidied 5% CO2 incubator at 37 C. After every 8 h, samples were drawn from
Surface charge density of each silk membrane was analyzed by measuring their three wells of each membrane-type and control plates by trypsinization using 0.25%
zeta potential value using cylindrical cell of electrokinetic analyzer with Ag/AgCl disc trypsin (Gibco, USA). At each sampling instant viable cell densities were measured
electrodes (Anton Paar GmbH, Graz, Austria). For each measurement, an irregular in triplicates.
plug of each silk membrane was placed between the two electrodes and the mea- Along with this, for visualization of attached adherent HT1080 cells on different
suring uid (0.001 mol/L KCl solution) was passed through them. For all the silk silk membranes, their scanning electron micrographs were also captured after 72 h
membranes, three zeta potential values were measured and the mean value was using the Evo50 scanning electron microscope (Zeiss, UK).
calculated. The pH was maintained by addition of 0.1 mol/L KOH or 0.1 mol/L HCl
solution. 2.3.5. Cell proliferation supporting nature of silk membranes
Proliferation of mammalian cells in the presence of three different silk mem-
2.3.3. Pore size branes (Seri-DSS, Seri-FSS and BF27PV taken separately) was tested by culturing
Pore size of each silk membrane was measured in triplicates using their three non-adherent HB8696 hybridoma cells on them in 24-well plates in exactly
scanning electron micrographs and IMAGE-J software (National Institutes of Health, the same manner as described in Section 2.3.4. Growth medium used was
USA, version 1.41). The images were captured using three strips of each silk mem- Hybricare 46-X medium supplemented with 20% heat inactivated FBS and 5%
brane (5 mm 5 mm each) by scanning electron microscope (Evo50, Zeiss, UK). penicillinstreptomycin solution. The seeding density of 1.4 105 cells/mL was used
46 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451

Fig. 2. Water contact angle measurement of Seri-DSS silk membrane by contact angle goniometer. (A) Contact angle of upper side of membrane. (B) Contact angle of lower
side of membrane.

for each well. All the plates were incubated in 5% CO2 incubator at 37 C. Here also, for high MAb productivity was determined by continuous perfusion experiments
three sets of these experiments were performed and after every 8 h sampling was of HB8696 cells in triplicates in perfusion bioreactor (Biostat, B.Braun, Germany)
done from three wells of each membrane-type and control plates by collection of at four different perfusion rates of 0.625 mL min1 , 0.75 mL min1 , 1.125 mL min1
suspended medium from them. At each sampling instant, viable cell densities were and 1.5 mL min1 under the same set of conditions as mentioned in Section 2.6.
measured in triplicates. Perfusion operation was started from 40 h, using two peristaltic pumps oper-
ated at equal ow rates. The growth medium used was Hybricare 46-X medium
2.4. Determination of critical shear stress of HB8696 hybridoma cells supplemented with 20% FBS and 5% penicillinstreptomycin solution. Working
volume of reactor was 750 mL. The seeding density of 3.2 105 cells/mL was
For operation of bioreactor below critical shear stress of HB8696 hybridoma used in each experiment. Each set of experiment was performed in triplicates.
cells, the optimum spinlter rotation speed was determined from the batch Samples were drawn after every 8 h, and at each sampling instant viable cell
experiments performed in triplicates in Biostat B.Braun bioreactor. Growth densities and MAb concentrations were measured in triplicates.
medium used was Hybricare 46-X medium supplemented with 20% FBS and 5%
penicillinstreptomycin solution. The initial spinlter rotation speed was set at 2.8. Analytical methods
10 rpm and then stepwise increase in spinlter rotation speed was done after every
5 h by a factor of 5 rpm. The pH inside the bioreactor was maintained at 7.4 with Cell density (viable and dead) was measured by trypan blue assay using a
the combined addition of 0.1 M sodium bicarbonate (NaHCO3 ; Merck, Germany) haemocytometer. Glucose concentration was measured by Glucose kit (Biolab diag-
and introducing 5% CO2 into the headspace of the reactor. The cultivation temper- nostics). Glutamine concentration was measured by enzymatic assay [22]. Lactate
ature was maintained at 37 C. Pluronic F-68 (0.1%, w/v, SigmaAldrich Corp., MO) concentration was measured by d-lactate colorimetric assay kit (Catalogue.K667-
was added to the medium as antifoaming agent, when required. The dissolve oxy- 100, Bio-vision, USA). Ammonia concentration was measured by Nesslers reagent
gen (DO) concentration was maintained at 50% of air saturation with the control [23]. LDH was measured by LDH cytotoxicity assay kit (Item no.10008882, Cayman
module of the reactor by adjusting the air/O2 /N2 ratio of the inlet gas. Working vol- chemical company, USA). MAb content was analyzed by Indirect ELISA and concen-
ume of the bioreactor was 750 mL. The seeding density used for each experiment trations were calculated by the standard curve of puried IgG1 . MAb was puried
was 3.0 105 cells/mL. Sampling was done after every 5 h and at each sampling from the culture supernatant by protein-G afnity column chromatography using
instant viable cell density and lactate dehydrogenase (LDH) concentration inside IgG purication kit (Bangalore Genie, India).
the bioreactor [21] was measured in triplicates to study shear effect on the cells.
2.9. Statistical analysis
2.5. Growth prole of HB8696 hybridoma cells in batch culture
All the data has been presented as the mean of three independent series of exper-
The growth prole of HB8696 hybridoma cells was studied by performing iments, each with n = 3 per group, mean standard deviation. Analysis of variance
their batch culture experiments in triplicates in Biostat B.Braun bioreac- (ANOVA) was performed using Stat-Ease Inc. software (USA).
tor using the Hybricare 46-X medium supplemented with 20% FBS and 5%
penicillinstreptomycin solution. Operating conditions were pH 7.2, temperature
3. Results and discussions
37 C and DO level 50%. Antifoaming agent was added, when required. The seeding
density used was 3 105 cells/mL. Spinlter rotation speed was set at 45 rpm (opti-
mized value). Samples were drawn after every 8 h and at each sampling instant, the In this section, the characterization of silk as spinlter screen
measurement of each variable (viable cell density, total cell density, percentage of for clog-free extended perfusion operation of hybridoma cells
viable and dead cells, and glucose, glutamine, lactate, ammonium, LDH and MAb and optimized perfusion strategy for enhancing MAb productiv-
concentrations) was carried out in triplicates.
ity against breast cancer using novel silk spinlter are described as
2.6. Comparative perfusion experiments in silk spinlter and stainless-steel
follows.
spinlter
3.1. Characterization of non-woven Bombyx mori silk as spinlter
Comparative continuous perfusion experiments of HB8696 hybridoma cells screen
were performed in perfusion bioreactor (Biostat, B.Braun, Germany) in triplicates
using Stainless-steel spinlter (pore size 20 m) and silk spinlter. The operating
conditions were maintained at pH of 7.2, cultivation temperature of 37 C and DO For selection of suitable silk as lter screen of the reusable spin-
concentration at 50% of air saturation. Antifoaming agent, Pluronic F-68 was used, lter module, the surface properties of three different non-woven
when required. Working volume of reactor was 750 mL. The seeding density used Bombyx mori silks, Seri-DSS, Seri-FSS and BF27PV and their effect
was 3.2 105 cells/mL. Spinlter rotation speed was set at 45 rpm (optimized value).
on cell adhesion and proliferation were studied. It was reported
Perfusion operation was started from 40 h, when viable cell density was started to
decrease in batch culture, using two peristaltic pumps at equal perfusion rate of that neutral or negatively charged hydrophobic membranes hav-
0.75 mL min1 . Sampling during these experiments was done after every 8 h and ing pore size ranges between 10 and 20 m were less prone to
at each sampling instant viable cell density, total cell density and MAb production clog compared to the positively charged hydrophilic membranes
were measured as the primary parameters for evaluating the performance while [9,10,1316]. Thus, among the three different silks, the most neg-
percentage retention of viable and dead cells, lactate, ammonium and LDH accumu-
lation inside the bioreactor were measured to correlate the changes in the medium
atively charged and hydrophobic one was selected as the suitable
affecting viability and productivity. At each sampling instant all of these parameters spinlter screen.
were measured in triplicates. A comparison of surface properties (Table 1) showed that Seri-
DSS was the most hydrophobic (water contact angle: 107.7 1.1
2.7. Perfusion experiments at different perfusion rates for the upper side and 102 2.7 for lower side, Fig. 2) and nega-
To enhance the MAb productivity from hybridoma cells, the performance of
tively charged (zeta potential value 38 0.5 mV) silk. Therefore,
silk spinlter at different perfusion rates was studied. The spinlter rotation speed Seri-DSS silk did not support the attachment and growth of adher-
was kept constant at an optimized value of 45 rpm. The optimized perfusion rate ent HT1080 cells on its surface over the entire culture period
 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451 47

Table 1
Comparison of properties of Seri-FSS, Seri-DSS and BF27PV silk membranes.

Properties Seri-DSS silk Seri-FSS silk BF27PV silk

Source Bombyx mori Bombyx mori Bombyx mori

Cell adhesion Does not support cell adhesion Supports cell adhesion Support cell adhesion
Cell proliferation Maximum viable cell density of Maximum viable cell density of Maximum viable cell density of
1.1 0.1 106 cells/mL achieved at 1.0 0.1 106 cells/mL achieved at 1.0 0.1 106 cells/mL achieved at
40 h 40 h 40 h
Pore size 14.9 1.8 m 15.4 0.8 m 14.5 1.1 m
Surface charge density Negative Positive Positive
38 0.5 mV 2.0 0.2 mV 7.9 0.2 mV
Wettability Both sides hydrophobic One side hydrophobic 101.5 2.1 Both sides hydrophilic
Upper side 107.7 1.1 Other side hydrophilic 15.3 1.7 Upper side 11.7 1.0
Lower side 102 2.7 Lower side 11.1 1.3

Fig. 5. Time prole of viable cell density (106 cells/mL) of non-adherent HB8696
Fig. 3. Time prole of viable cell density (106 cells/mL) of adherent HT1080 cells in
cells in control wells and Seri-DSS, Seri-FSS, BF27PV silks containing wells of 24-well
control wells and Seri-DSS, Seri-FSS, Bf27PV silks containing wells of 24-well plates.
plates.

of 120 h, as shown in Fig. 3. Similarly, in the scanning electron 3.2. Batch culture for determination of critical shear stress of
micrographs also no cells were found to be attached on the Seri- HB8696 hybridoma cells
DSS silk after 72 h (Fig. 4). To rule out if non-attachment and
non-proliferation was caused by the Seri-DSS silk, non-adherent Batch culture of HB8696 hybridoma cells were performed in
HB8696 cells were also cultured over all the silk membranes. It bioreactor at different spinlter rotation speeds to determine the
was observed that the presence of circular pieces of silk mem- critical shear stress. From the experiments (Fig. 7), it was found that
branes had no detrimental effect on cell proliferation compared to initially with an stepwise increase in spinlter rotation speed from
the control (Fig. 5). Therefore, it was concluded that Seri-DSS silk 10 to 45 rpm, by a factor of 5 after every 5 h, the viable cell density
neither supported cell attachment nor adversely affected cell prolif- continuously increase up to 40 h and at 40 h, the maximum viable
eration. Further, its average pore diameter of 14.9 1.8 m (Fig. 6) cell density of 1.5 0.1 106 cells/mL was achieved. But after 40 h,
was also between 10 and 20 m, suitable for effective retention of due to further increase in spinlter rotation speed above 45 rpm,
hybridoma cells. viable cell density started to decreased and at 50 h, the decreased
Due to all of these features, Seri-DSS silk was used as the lter viable cell density of 0.6 0.1 106 cells/mL was achieved. This
screen of the designed spinlter module. shows that spinlter rotation speed above 45 rpm is detrimental for

Fig. 4. Scanning electron micrographs of silk membranes after 72 h of adherent HT-1080 cells culture. (a) Seri-DSS (b) Seri-FSS and (c) BF27PV silk.
48 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451

Fig. 6. Pore size distribution of Seri-DSS silk membrane. (AC) Scanning electron micrographs of three Seri-DSS silk membrane. (D) Pore size distribution curve of image A,
B and C measured by Image-J software.

HB8696 cells and above this, cell lysis occurred. Therefore after 40 h,
a sharp increase in LDH concentration up to 520 18.6 U/mL was
obtained. Therefore, for all the further batch and perfusion experi-
ments of HB8696 cells, the spinlter rotation speed of 45 rpm was
used.

3.3. Growth prole of HB8696 hybridoma cells in batch culture

The growth prole of HB8696 hybridoma cells during batch

culture was studied (Fig. 8). The initial concentration of glu-
cose and glutamine were 5.3 mM and 5.4 mM respectively. From
the gures, it was found that viable cell density was increased
up to 40 h and at 40 h; the maximum viable cell density of
1.5 0.1 106 cells/mL was achieved. Beyond this, viable cell den-
sity started to decrease gradually due to the accumulation of
lactate concentration above 23.3 0.8 mM and ammonium con-
centration above 5.4 0.3 mM, as observed in all the three batch
experiments [20,2426]. However, MAb production was contin-
Fig. 7. Effect of different spinlter rotation speeds on HB8696 hybridoma cells dur-
ing batch culture in bioreactor. Here vcd is viable cell density (106 cells/mL) and ued and the maximum MAb concentration of 286.2 41.3 mg/L
LDH is lactate dehydrogenase accumulation (U/mL). was achieved at 96 h. Beyond 96 h, due to further accumulation
 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451 49

Fig. 8. Time proles of cell growth and product formation of non-adherent HB8696
Fig. 9. Comparative time proles of cell growth and product formation of non-
cells during batch culture in Biostat B.Braun bioreactor. (A) States characterizing
adherent HB8696 cells during perfusion culture in bioreactor using stainless steel
the growth of HB8696 cells. Here vcd is viable cell density (106 cells/mL); tcd is
(SS) and silk spinlter. (A) States characterizing the growth of HB8696 cells. Here
total cell density (106 cells/mL); %v is cell viability (%). (B) States characterizing
vcd is viable cell density (107 cells/mL); tcd is total cell density (107 cells/mL);
the nutrient consumption and waste products accumulation by HB8696 cells. Here
%v is cell viability (%). (B) States characterizing waste products accumulation by
Glu is glucose concentration (mM); Gln is glutamine concentration (mM); Lac is
HB8696 cells. Here LC is lactate concentration (mM); AC is ammonium concentra-
lactate concentration (mM); Amm is ammonium concentration (mM). (C) States
tion (mM). (C) States characterizing product formation of HB8696 cells. Here MAb
characterizing product formation of HB8696 cells. Here MAb is monoclonal antibody
is monoclonal antibody concentration (mg/L); %v is percentage of viable cells; %d is
concentration (mg/L); %v is percentage of viable cells; %d is percentage of dead cells;
percentage of dead cells; LDH is lactate dehydrogenase concentration (U/mL).
LDH is lactate dehydrogenase concentration (U/mL).

From these results, it was concluded that to increase the MAb

of lactate above 30 2.4 mM and ammonium above 9.2 0.2 mM, productivity, perfusion operation should be started from 40 h, so
a sharp decrease in viable cell density occurred marked with an that the onset of inhibitory concentrations of lactate and ammo-
increase in LDH concentration and commensurate decrease in MAb nium could be delayed and the duration of cell growth phase and
concentration. production phase extended.
50 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451

Table 2
Comparative viable cell densities and monoclonal antibody productivities of HB8696 cells during batch culture in bioreactor and perfusion culture using stainless steel
spinlter and silk spinlter.

HB8696 hybridoma cells

Parameters Time (h) Bioreactor Stainless steel spinlter Silk spinlter

Batch Perfusion

Viable cell density (107 cells/mL) 40 0.15 0.1 0.5 0.1 0.9 0.1
96 0.07 0.1 1.3 0.1 2.1 0.1
152 1.1 0.1 2.5 0.1
Monoclonal antibody productivity (g L1 day1 ) 0.53 0.94 1.48
Total monoclonal antibody produced (g) 2.6 9.4 14.8
Increase in productivity from stainless steel to silk 57.4%

Values shown in bold are the maximum viable cell density achieved in each case.

Fig. 10. Time proles of cell growth and product formation of non-adherent HB8696 hybridoma cells at different perfusion rates and constant spinlter rotation speed of
45 rpm during perfusion culture using silk spinlter. (A) Viable cell density (107 cells/mL). (B) Monoclonal antibody concentration (mg/L).

3.4. Comparative study in silk spinlter and stainless-steel reached 56 h prior than the silk spinlter and thus the maximum
spinlter viable cell density of only 1.3 0.1 107 cells/mL was achieved.
Similarly, the second inhibitory level of lactate (>30 mM) and
This comparative study demonstrated the application of silk ammonium (>9.2 mM) crossed 40 h earlier as compared to the silk
spinlter for clog-free extended perfusion operation of hybridoma spinlter, therefore the MAb concentration as well as MAb produc-
cells (Fig. 9). It is clear from the gure that silk spinlter provides tivity of only 528.6 24.2 mg/L and 0.94 g L1 day1 respectively
the extended cell growth phase of 152 h and extended MAb pro- was achieved.
duction phase of 192 h, compared to the cell growth phase of 96 h Therefore, it can be concluded that silk spinlter performs much
and MAb production phase of 152 h achieved using stainless-steel better than the Stainless-steel spinlter (Table 2) and provides
spinlter. 57.4% increase in MAb productivity.
Using silk spinlter, a maximum viable cell density of This is due to the lower likelihood of hydrophobic and negatively
2.5 0.1 107 cells/mL was achieved at 152 h. After this, the viable charged Seri-DSS silk screen of the silk spinlter to clog with nega-
cell density start to decrease gradually due to the accumulation of tively charged cells and cell debris as compared to the hydrophilic
lactate above 23.5 mM and ammonium above 5.4 mM, as observed and positively charged stainless steel screen of stainless steel spin-
at 40 h during batch culture of HB8696 cells. However, the produc- lter. Due to the lower clogging tendency of silk spinlter, while
tion of MAb was still continued and at 192 h, the maximum MAb using it during the perfusion culture, the accumulation of inhibitory
concentration of 723.9 70.5 mg/L was achieved. The overall MAb concentrations of toxic metabolites (lactate and ammonium)
productivity was 1.48 g L1 day1 . inside the bioreactor was delayed and thus the healthy environ-
In comparison, when the Stainless-steel spinlter was used, the ment for cell growth was maintained for longer period of time
rst inhibitory level of lactate (23.5 mM) and ammonium (5.4 mM) [2026]. Therefore it provides extended duration of cell growth and

Table 3
Comparative maximum viable cell densities and maximum monoclonal antibody concentrations achieved during perfusion culture of HB8696 hybridoma cells using silk
spinlter at different perfusion rates.

S. no. Perfusion rate Rotation speed Maximum viable cell Time2 (h) Maximum monoclonal antibody Time4 (h)
(mL min1 ) (rpm) density1 (107 cells/mL) concentration3 (mg/L)

1 0.625 45 2.3 0.1 128 454 25.5 152

2 0.75 45 2.5 0.1 152 723.9 70.5 192
3 1.125 45 3.5 0.1 176 793 22.2 200
4 1.5 45 2.1 0.0 120 432 22.2 152
a
The maximum viable cell density 1 (mean standard deviation) occurs at time 2.
b
The maximum monoclonal antibody concentration 3 (mean standard deviation) occurs at time 4.
 S. Kamthan et al. / Enzyme and Microbial Technology 6465 (2014) 4451 51

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