PROTEIN DRUG

DEVELOPMENT

DR HENNY RACHDIATI
LECTURE OUTLINE
 Introduction
 Preparation for Protein Formulation Development
 Pre formulation Development
• Accelerated Stability Studies
• Development of Analytical Method
• Evaluation of the Significance of Problem
 Formulation Development
LEARNING OUTCOME
 Identify the importance protein formulation development
 Describe how to approaches protein formulation development
INTRODUCTION
 PROTEIN
 Contain amine (NH2) and carboxyl group (COOH)
 The building blocks of proteins are amino acid (monomers)
 Amino acids are connected by a special type of bond called a
peptide bond
 Amino acid chains are called are polypeptide
 A protein contains one or more polypeptide chains
 EACH PROTEIN HAS A PERSONALITY
 A protein’s personality contributes to physicochemical
attributes and final drug product formulation but is
difficult to guess and/or fix without conducting
systematic research.
 When a new protein is in the development phase, one
seeks the most effective and efficient process to
identify a formulation given the resource, budget, and
timeline.
PREPARATION FOR
PROTEIN FORMULATION DEVELOPMENT
FORMULATION DEVELOPMENT
 Critical steps in developing a protein as a therapeutic product
 Ideal to be able to develop a pure pharmaceutical containing only the
native protein.
 In general, commercial therapeutic protein formulations are developed
under the assumption that some degree of physicochemical changes will
occur during storage and handling.
 Developing commercial formulations requires a clear understanding of the
potential market.
Resource Requirements For Initial Protein Formulation Development

Resources Requirement Example

Representative of manufacturing process;
sufficient quantity to cover dose bracket, Purified bulk,
formulation variables, and stress conditions; sample from final
Purified protein
minimum complication by impurity purification
(precipitation of impurity, degradation by process
impurity like proteolytic cleavage).
Pharmaceutically acceptable quality,
manufacturers with qualified production
Qualified procedures and sufficient scale, specifications
USP, Ph. Eur, JP
excipients on critical impurities, quality that can be
carried on to clinical studies and commercial
distribution.
Resource Requirements For Initial Protein Formulation Development
Resources Requirement Example
Capability to sterilize container/closure
Sterile hood,
Access to fill components; fill/finish under aseptic
filling machine,
finish facility environment; head-space purge system;
lyophilizer
drying equipment
CD, UV,
Structural analyses; concentration
fluorescence,
Analytical determination,
HPLCs, mass-
Instrument chromatographic analyses; electrophoresis;
spectrometry,
bioassays; other micro characterizations.
SDS-PAGE
Freezer,
Controlled temperature, controlled light refrigerator,
Facility to
exposure, incubator, light
accommodate
controlled relative humidity, devices to chamber,
stability studies
provide controlled agitation. RH Incubator,
agitator
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PREFORMULATION
DEVELOPMENT
 PREFORMULATION DEVELOPMENT
Studies are designed to learn about the protein’s susceptibility
to a variety of pharmaceutically relevant stresses.

 general characterization of the product

 investigation of potential stability issues
 development of relevant analytical methods
 establishment of a stability profile with stability-indicating assays
 identification of major formulation challenges
 Examples of Information Useful for Designing Formulation Studies
Information Examples
Clinical indication Site of treatment (self-administration, office visit, hospital), methods of delivery,
concomitant medication, competition.

Patient population Age, strength, tolerability, capability to manipulate devices,

sensitivity to excipient
Typical routes of delivery Injectables (IV, SC, IM, IP, ICV, IT, IO), topical, inhalation, nasal, oral, etc.

Dose requirement PK profile, frequency of dosing, variable vs. fixed dose, single- dose/multidose

Drug interaction Co-administration with other drug, dilution or reconstitution with other solution;
presence of undesirable compounds like reducing sugars, preservatives

Typical dosage forms Liquid, lyophilized, spray-dried, aerosol by liquid or powder, other novel carrier;
stability, physical properties, reconstitution art
Container/closure Vial/stoppers, prefilled syringes, prefilled cartridges, dual chamber cartridges,
blister packages, product contact material, leacheates, breakage, light sensitivity,
moisture penetration
Delivery device Syringes, prefilled-syringes, pen injectors, auto-injectors, needle-free injectors,
inhalation devices, infusion pumps
Information obtained from Pre-formulation Studies for Pharmaceuticals

Characterization Examples
Physical properties Primary, secondary, tertiary and quaternary structures, solubility, viscosity, self-
association, hydrophobicity, molecular weight, extinction coefficient,
glycosylation, effects of ionic strength, etc.

Biological properties Substrate or receptor affinity, in vitro activity model, in vivo preclinical model, etc.
 Various Conditions Used To Accelerate Protein Degradation
Stresses Routine Ranges Practical Application Problems to Monitor
Temperature 0-50°C Storage, shipping, Structural changes (precipitation,
handling, delivery aggregation, recovery loss),solubility,
increased reaction rates for all degradations

Light >1.2 million lux hrs Light exposure, Oxidation, cleavage

illumination, container, package
> 200 watt hrs/square
meter UV energy

Freezing Multiple freeze-thaw, Frozen storage, Precipitation, aggregation, pH change,

liquid nitrogen freeze accidental freezing, crystallization of excipients
lyophilization

Oxidation Oxygen purge, peroxide Storage, excipient Oxidations, inactivation

spike stability, impurity
Humidity 0-100% RH Storage, container Moisture content, moisture related
integrity, powder degradations
Mechanical Vortex, agitation, shear Manufacturing, filling, Precipitation, aggregation,
stress (3000 s-1) shipping, handling, recovery loss
delivery
Other Impurities, pH, Precipitation, aggregation,
denaturans denaturing excipients recovery loss, structural changes
Typical Methods Used to Characterize Proteins and Degradation Products
Methods Example Application
HPLC, FPLC, low pressure LC; sizeexclusion, Most physical and
Column reversed-phase, ion-exchange, hydrophobic, affinity chemical degradations,
chromatography columns; coupled with UV, fluorescence, RI, and other excipient impurities,
analytical instruments as detectors leacheates
Degradations with
SDS-PAGE, native PAGE, isoelectric
Electrophoresis changes in size and/or
focusing, capillary electrophoresis, etc.
charge
Structural changes,
Spectroscopy CD, fluorescence, FTIR, UV, Raman, NMR, etc. chemical modifications of
side groups
Protein structure,
Differential scanning calorimetry, thermo gravimetric
Thermal Analysis lyophilized cake structure,
analysis, thermomechanical analysis, etc.
powder characterizations
Aggregation, precipitation,
Light scattering/ Dynamic light scattering, other light scattering devices,
molecular weight
turbidity turbidity, particle size determination, particle counter, etc.
determination
Identification of impurities
Other micro Peptide mapping, peptide sequencing, amino acid and chemical degradation,
characterization analysis, mass spectrometry, other specific analyses for analysis of complex
methods individual reactive groups proteins, e.g., antibody
and glycoprotein
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FORMULATION
DEVELOPMENT
• LIQUID FORMULATIONS
• SOLID DOSAGE FORMS
• SINGLE DOSE AND MULTIDOSE FORMS
Formulation Options for Protein Pharmaceuticals

Different types of formulations need to be developed based on :

 clinical needs
 patient compliance
 delivery method
 stability of the drug
 storage and distribution
 market competitiveness
LIQUID FORMULATION

 Generally preferred due to the convenience of manufacturing and use.

 Protein drugs may not be stable enough.
DRIED /SUSPENSION FORMULATION

 Dried formulations (e.g., lyophilized) or suspension formulations

(e.g., insulin zinc suspension) have been successfully used to
overcome stability problems.
 In addition, specific applications and delivery may demand the
appropriate type of formulation, e.g., a spray-dried powder for
pulmonary delivery.
 Typical Stability Problems Observed in Protein Pharmaceuticals.
Problem Potential causes Possible solutions
Non-covalent Solubility, structural changes, heat, pH, ionic additives, amino acids,
aggregation shear, surface, denaturants, impurities surfactants, protein concentration,
raw material purity
Covalent aggregation Disulfide scrambling, other unknown pH, inhibit non-covalent
mechanisms aggregation
Deamidation pH < 5.0 or pH >6.0 pH optimization

Cyclic imide pH around 5 pH optimization

Cleavages Protease impurity, other unknown pH, product purity, inhibitors

mechanisms
Oxidation Active oxygen species, free radicals, Excipient purity, free-radical
metals, light, impurity scavenger, active oxygen
scavengers, methionine
Surface denaturation, Low protein concentration, specific Surfactants, protein concentration,
adsorption affinity, protein hydrophobicity pH
 Important Components of Protein Formulations

Formulation Desired Atributes Examples

Variables
pH Provides good physical properties of
protein, minimize degradations
Stabilizer Inhibit degradations, effective at low Surfactants, sugars, salts,
concentrations antioxidants
Solubilizer Improve the solubility, effective at low Salts, amino acids, surfactants
concentrations
Buffer Good buffering capacity, stable to Phosphate, acetate, histidine,
temperature change, stable to freezing, glutamate
good safety record
Tonicity modifier Inert, good safety record Sodium chloride, sorbitol,
bulking agent mannitol, glycine
Design of Study
 Study title
 Study objective
 Source and quality of drug substance and excipients
 Material preparation
 Formulation matrix
 Formulation variables
 Protein concentrations or bracket
 Analytical methods
 Storage conditions (temperature, light, humidity)
 Additional sample handling conditions (temporary exposure to stresses, container
orientation, etc.)
 Sampling schedule and expected duration of the study
 Plan for data analysis and report
Necessary Studies for Formulation Development
 Storage Stability Study : In general, a shelf life of 18 month is considered
acceptable for commercialization
 Process Development : Formulations that can be prepared on a small scale
without experiencing any problems may encounter significant problems
during the scale-up of the process.
 Transportation, Handling and Delivery Study : Unexpected environmental
changes can be encountered during the distribution and handling of products
(e.g., accidental freezing, exposure to temperatures different from the
recommended conditions,vigorous agitation, etc.)
 Preclinical and Clinical Studies : Results documenting the maintenance of
the biophysical and biochemical properties of the protein are essential for a
final formulation decision.
Strategies to Overcome Difficult Formulation Problems

 Qualification of Degradation Products

 Site-directed Mutagenesis to Improve Properties
 Chemical Modifications
 Unconventional Dosage Forms
FORMULATION IN COMMERCIAL
PRODUCT DEVELOPMENT
 FORMULATION IN COMMERCIAL PRODUCT
DEVELOPMENT
 Critical Formulation Decisions During Pharmaceutical Development
 Formulation for Early Preclinical and Clinical Studies
 Commercial Formulation
 Regulatory Issues in Formulation Development
 Guidelines for Stability Studies
 Results Required to Apply for Regulatory License for a Drug
Product
 Results Required to File Formulation Amendments
REFERENCES
 Chang, B.S. and Hershenson, S. 2002. Practical approaches to protein
formulation development. in "Rationale Design of stable protein
formulations-theory and practice“ (J.F. Carpenter and M.C.
Manning eds.) Kluwer Academic/Plenum publishers, New York,
 Daan J.A. Crommellin et al, 2008. Pharmaceutical Biotechnology
“Fundamental and Applications, Informa Healthcare, London UK
 Pearlman, R. and Wang,Y.J., 1996. Formulation, characterization,
and stability of protein drugs: case histories. Pharm. Biotech. Ser.
Volume 9. Plenum Press, N.Y.
THANK YOU