1

Practical Approaches to Protein

Formulation Development

Byeong S. Chang and Susan Hershenson

INTRODUCTION

As is the case with other pharmaceuticals, formulation development is one of the

critical steps in developing a protein as a therapeutic product. Development of
stable protein formulations may require even more resources and effort than con-
ventional small molecule pharmaceuticals. Proteins typically have more stability
issues as a result of their complexity and delicate structural stability. Fortunately,
a great deal of research regarding protein stability has been conducted and this
information is readily available in the literature (reviewed by Manning et aI., 1989;
Chen, 1992; Ahem and Manning, 1992a, 1992b; Arakawa et al., 1993; Cleland
et aI., 1993; Wang and Pearlman, 1993; Pearlman and Wang, 1996; Volkin and
Middaugh, 1997). Ultimately, it would be ideal to be able to develop a pure phar-
maceutical containing only the native protein. However, it is not practical to have
only the native form of a protein in the formulation because the protein must be
purified from a complex biological mixture containing a pool of other proteins
which includes misfolded, denatured, and degraded forms of the same protein.
Furthermore, a major challenge is to maintain the integrity of the purified protein
during routine pharmaceutical processing, storage, handling, and delivery to the
patient. One could envision achieving this goal by developing a formulation with

Byeong S. Chang and Susan Hershenson • Department of Pharmaceutics and Drug Delivery,
Amgen, Inc., Thousand Oaks, CA 90132.
Rational Design of Stable Protein Formulations, edited by Carpenter and Manning. Kluwer Academic I Plennrn
Publishers, New York, 2002.

J. F. Carpenter et al. (eds.), Rational Design of Stable Protein Formulations

© Kluwer Academic/Plenum Publishers, New York 2002
2 Byeong S. Chang and Susan Hershenson

perfect stability, i.e., no physical and chemical change in the protein. Because pro-
teins are complex molecules composed of numerous reactive chemical groups and
delicate three-dimensional structures, identifying a set of conditions to keep all
components stable is virtually impossible. In general, commercial therapeutic
protein formulations are developed under the assumption that some degree of
physicochemical changes will occur during storage and handling.
Realizing that it is impossible to develop a perfectly stable formulation,
especially while meeting an aggressive product development timeline, the main
objective then becomes one of maintaining the appropriate safety and efficacy of
the product. In order to achieve this objective, it is imperative to understand the
broad spectrum of degradation pathways affecting proteins, and to have available
equipment and expertise in an extensive repertoire of analytical methods. For-
mulation development focuses on determining the potential degradation path-
ways, assessing the significance of each and optimizing variables to minimize the
degradation products that are clinically significant.
Regulatory guidelines also are critical elements for guiding formulation
development. They provide information about how to conduct studies and obtain
useful results for evaluating formulations. The results obtained allow formulation
scientists to write an appropriate developmental pharmaceutics section in regu-
latory filings. The guidelines also help to evaluate the significance of some
inevitable degradation products that are produced during manufacturing, shipping
and storage. For example, if the degradation products have properties compara-
ble to those of the desired product with respect to activity, efficacy, and safety,
they can be classified as product-related substances. This classification is signif-
icantly different from considering the degradation product as an impurity when
there is not sufficient supporting evidence to justify classification as a product-
related substance (Appendix, Regulatory Document 1). Understanding of these
practical issues of regulatory requirements is critical for formulation scientists
during design, implementation, evaluation and reporting of their studies.
In addition to insight into the scientific and regulatory issues, developing
commercial formulations requires a clear understanding of the potential market.
For example, indication, patients, method of delivery, frequency of dosing, typical
dose requirement, market distribution and other business-related information will
provide directions for the design of a successful formulation. Also, it is impor-
tant to consider the competitiveness of the formulation as compared to other
products available in the market.
In this chapter, an overview of critical factors affecting the design of thera-
peutic protein formulations and a general guide to developing commercially
viable dosage forms for protein pharmaceuticals will be discussed. Since the
majority of practical issues are not covered very well in the scientific literature,
this chapter also includes information from regulatory guidance documents (see
Appendix), labels from marketed products and routine industrial experience.
Practical Approaches to Protein Formulation Development 3

PREPARATION FOR FORMULATION DEVELOPMENT

Resource Requirements for Formulation Development

A list of resources that should be available before starting formulation devel-

opment is summarized in Table 1. Some resources may not be as critical as others,
depending on the nature of problems encountered, but it is important to have suf-
ficient resources to discover major formulation issues as early as possible in the
product development process. Without the appropriate equipment and personnel,
the development of an acceptable formulation can be greatly hampered, poten-
tially to such a degree that the product is never brought to market. Of course, not
all of the resources need to be at the company developing the therapeutic pro-
tein, but if outside contractors are employed it is essential that timely access
to resources is available. In addition, the quality of raw materials should be
carefully evaluated because unexpected impurities may introduce unnecessary

Table 1.
Resource Requirements for Initial Protein Formulation Development
Resources Requirement Example

Purified protein Representative of manufacturing process; Purified bulk, sample

sufficient quantity to cover dose bracket, from final
formulation variables, and stress conditions; purification process
minimum complication by impurity
(precipitation of impurity, degradation by
impurity like proteolytic cleavage).
Qualified Pharmaceutically acceptable quality, manufacturers USP, Ph. Eur, JP
excipients with qualified production procedures and
sufficient scale, specifications on critical
impurities, quality that can be carried on to
clinical studies and commercial distribution.
Access to fill Capability to sterilize container/closure Sterile hood,
finish facility components; fill/finish under aseptic filling machine,
environment; head-space purge system; lyophilizer
drying equipment
Analytical Structural analyses; concentration determination, CD,UV,
instruments chromatographic analyses; electrophoresis; fluorescence, HPLCs,
bioassays; other microcharacterizations. mass-spectrometry,
SDS-PAGE
Facility to Controlled temperature, controlled light exposure, Freezer, refrigerator,
accommodate controlled relative humidity. devices to provide incubator, light
stability studies controlled agitation. chamber, RH
incubator. agitator
4 Byeong S. Chang and Susan Hershenson

Table 2.
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, 1M, IP, ICV, IT, 10), 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

complications in the stability profile that is determined during formulation devel-

opment and testing.

Useful Information for Designing Formulations

The configuration of a protein formulation is affected by how the drug

will be used as a product. If such information is available when designing for-
mulation studies, it is recommended to consider the limitations and challenges
associated with each application. Examples of such information are listed in
Table 2.

PREFORMULATION DEVELOPMENT

It is important to understand the critical properties of a protein before start-

ing large studies to design and test the final formulation. Preformulation studies
are designed to learn about the protein's susceptibility to a variety of pharma-
ceutically relevant stresses. The main objectives of preformulation research
Practical Approaches to Protein Formulation Development 5

Table 3.
Information Obtained from Pre-formulation Studies for Protein 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.

include: general characterization of the product; investigation of potential stabil-

ity issues; development of relevant analytical methods; establishment of a
stability profile with stability-indicating assays; and identification of major
formulation challenges. A summary of these issues will be presented in this
section.

Characterization of Protein Pharmaceuticals

There is available in the literature extensive coverage of analytical methods

and their principles for the characterization of proteins (e.g., Jones, 1993; Reub-
saet et aI., 1998; Herron et aI., 1995). General points to consider when charac-
terizing a protein as an active ingredient are listed in Table 3. As a protein's
biological activity is dependent on its structure, significant emphasis has been
given to structural properties and stability against various stresses.

Accelerated Stability Studies

In order to predict potential stability problems within a short period of time

and to develop appropriate analytical methods, proteins are exposed to stronger-
than-real stresses and various degradation products induced by the stresses are
examined. The results obtained from these so-called "accelerated stability
studies" might also be useful to predict the kinetics of the degradation processes
under real handling conditions, when there are not sufficient real-time results
available because of time and resource constraints. However, the accelerated sta-
bility study is not acceptable to determine expiry of the product, so it is best used
to rank order the importance of different degradation pathways. Approaches and
cautions in the extrapolation of data from accelerated stability testing to real-time
stability and normal handling conditions are discussed in detail below.
6 Byeong S. Chang and Susan Hershenson

Table 4.
Various Conditions Used to Accelerate Protein Degradation
Practical
Stresses Routine ranges applications 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
liquid nitrogen freeze accidental freezing, change, crystallization of
lyophilization excipients
Oxidation Oxygen purge, peroxide Storage, excipient Oxidations, inactivation
spike stability, impurity
Humidity 0-100% RH Storage, container Moisture content, moisture
integrity, powder related degradations
Mechanical Vortex, agitation, shear- Manufacturing, Precipitation, aggregation,
stresses stress (3000s- l ) filling, shipping, recovery loss
handling, delivery
Other Impurities, pH, Precipitation, aggregation,
denaturants denaturing recovery loss, structural
excipients changes

Conditions to accelerate various degradation reactions in protein products

and potential problems to monitor are listed in Table 4. Proteins contain numer-
ous amino acid side chains and delicate three-dimensional structures, which can
be susceptible to different stresses. Therefore, it is important to test the protein
under a variety of physical and chemical stresses in order to provide a good sim-
ulation of the degradation products that can be generated.

Development of Analytical Methods

A brief summary of typical analytical methods is presented in Table 5. It is

essential to have a wide range of analytical methods available to identify and
characterize degradation products. The analytical methods should be further
selected and customized to accommodate the specific needs for each protein
product.
Practical Approaches to Protein Formulation Development 7

Table 5.
Typical Methods Used to Characterize Proteins and Degradation Products
Methods Examples Applications

Column chromatography HPLC, FPLC, low pressure LC; size- Most physical and
exclusion, reversed-phase, chemical degradations,
ion-exchange, hydrophobic, affinity excipient impurities,
columns; coupled with uv, leacheates
fluorescence, RI, and other
analytical instruments as detectors
Electrophoresis SDS-PAGE, native PAGE, isoelectric Degradations with changes
focusing, capillary electrophoresis, in size and/or charge
etc.
Spectroscopy CD, fluorescence, FTIR, UV, Raman, Structural changes,
NMR, etc. chemical modifications
of side groups
Thermal analysis Differential scanning calorimetry, Protein structure,
thermogravimetric analysis, lyophilized cake
thermomechanical analysis, etc. structure, powder
characterizations
Light scattering/turbidity Dynamic light scattering, other light Aggregation, precipitation,
scattering devices, turbidity, particle molecular weight
size determination, particle counter, determination
etc.
Other Peptide mapping, peptide sequencing, Identification of impurities
microcharacterization amino acid analysis, mass and chemical
methods spectrometry, other specific analyses degradation, analysis of
for individual reactive groups complex proteins, e.g.,
antibody and
glycoprotein

Evaluation of the Significance of Problems

As stated earlier, it would be ideal to have a pure protein in an absolutely

stable formulation. In reality, however, scientists have to design formulation
based on compromises that deal with several different potential problems. To
make matters worse, the formulation needs to be recommended long before it is
evaluated fully, because of typical aggressive timelines in the industry. In general,
the formulation will have to be optimized based on assumptions and extrapola-
tions of results obtained during a limited time. Therefore, it is important to utilize
the given time efficiently, by collecting as much relevant information as possible
for evaluating the significance of each problem.
Due to the marginal stability of proteins, it is possible to create rapidly a
variety of degradation products during accelerated stability testing. However, not
8 Byeong S. Chang and Susan Hershenson

all the degradation products that are observed will be significant under normal
handling, shipping and storage conditions. Furthermore, the rank order of differ-
ent degradation processes under accelerated stability testing will NOT be the
same under practical handling conditions, because each reaction has a different
temperature dependency, i.e., reaction order and activation energy. Another
important thing to keep in mind when evaluating different degradation processes
is the contribution to the pharmaceutical quality of the product. Critical degra-
dation products should be designated not on the quantity obtained during
accelerated stability studies, but on a comprehensive understanding of their
contributions to the quality of the product.
Quantitative Assessment. Usually, the rate of each degradation reaction
during real storage conditions (e.g., at 4-8°C) should be very slow. In order to
predict the rate under real storage conditions, reaction rates obtained under accel-
erated stability conditions can be extrapolated by using the Arrhenius equation.
Predicting the correct reaction rate requires a proper understanding of the
reaction order, because the amount of the degradation product does not linearly
increase over time unless the reaction follows zero-order kinetics. One way to
determine the reaction order is to calculate the linearity between the concentra-
tion of residual native protein and time. The reaction order can be calculated from:

dCjdt =kC (1)

where, C is the protein concentration, t is the time, k is the reaction rate constant,
n is the reaction order. For a Zero order reaction n = 0 and Co - C = k· t. For a
first order reaction n = I and log Co - log C = k· t. For a second order reaction n
= 2 and lIC - lICo = k· t. For each reaction order a characteristic transformation
of concentration will show a linear relationship with time, e.g., log C will show
a linear relationship with time for a first order reaction, whereas lICo will be linear
versus time for second order reactions.
After obtaining the reaction rate constants for different temperatures, the
activation energy can be found by using with the Arrhenius equation.

k = A·e(-Ea!RT) (2)

The activation energy is obtained by plotting (logk) vs (1rr) and determining the
slope of the plot. Using the activation energy, the reaction rate constant for
real storage condition, e.g., 2-8°C for refrigerated storage, can be estimated by
extrapolation.
Care must be taken when extrapolating the rate constants because there are
numerous cases when the Arrhenius relationship does not apply. The Arrhenius
reaction applies only to irreversible reactions where the product is accumulated
as a single quantifiable species. If the degradation product is the result of serial
Practical Approaches to Protein Formulation Development 9

reactions, the reaction order and rate constant will be determined only by the rate-
limiting reaction at the testing condition. If the rate-limiting reaction changes or
another significant variable is introduced at the extrapolated condition, then the
extrapolated rate constant will be in error. Such complications routinely can be
found in various degradation pathways that are affected by temperature-sensitive
reactions such as structural changes, pH changes, by physical changes such as
the glass transition of an amorphous phase in a lyophilized formulation and by
changes in reactants like dissolved oxygen. For example, proteins have relatively
high activation energies for structural changes. Thus, many reactions that are
dependent on a major perturbation of the native protein structure tend to cause
substantial damage under accelerated storage conditions, e.g., higher temperature.
However, many of these reactions are not necessarily major problems when the
product is stored at 2-8°C where the protein maintains its native conformation.
In contrast, degradation reactions with lower activation energies, which might not
be coupled to protein conformational changes (e.g., oxidation of surface methio-
nine residues), tend to be much more problematic in the development of protein
formulations.
Results from stresses (e.g., agitation) other than heat imposed in accelerated
stability studies potentially could be extrapolated in a similar way to storage and
actual handing conditions. The major challenge is the ability to assess quantita-
tively the magnitude of the stress so that the stress-stability relationship can be
established. To date there are not published guidelines for such quantitation.
Qualitative Assessment. Not all of the degradation products are equivalent
in terms of their contribution to the pharmaceutical quality of the protein product.
Therefore, a qualitative assessment of each degradation product is important
when weighing their significance. Regardless of their quantity, some degradation
products are generally less acceptable than others. If the degradation product
comprises the safety of the product, then it should be considered less acceptable.
For example, even at levels of a few percent or less of the total protein popula-
tion, non-native aggregates can cause adverse reactions in patients such as
immune responses and even anaphylactic shock. Also, if the degradation reaction
results in the inactivation of protein, it needs to be considered more important
than other degradation reactions that do not affect the activity. For example,
certain chemical changes (e.g., deamidation) may not alter the activity of a given
product, whereas other reactions (e.g., oxidation) may render that product inac-
tive. The relative impact of each degradation product on safety and efficacy
cannot be predicted and must be determined for each protein therapeutic.
Information useful for the qualitative evaluation of degradation products
includes the identity, clinical and preclinical experiences. with the degradation
product, biological activity, stability and potential side effects. Regulatory docu-
ments also provide clear guidance about what assessment is necessary for protein
degradation products (Appendix, Regulatory Documents 2,3). The criteria for
10 Byeong S. Chang and Susan Hershenson

identification, reporting and qualification of degradation products are set up based

on the total patient exposure and percentage of the degradation products (Appen-
dix, Regulatory Document 3). When certain degradation products cannot be pre-
vented from forming in the formulation, their inclusion in the product must be
qualified, which may require that additional information on safety and efficacy
be obtained as recommended in the regulatory guidelines (Appendix, Regulatory
Documents 2,3). Obtaining this information requires a significant amount of addi-
tional work, which may include clinical studies.

FORMULATION DEVELOPMENT

As discussed above, the critical parameters affecting the pharmaceutical

quality of a protein therapeutic are defined during the preformulation studies. In
formulation development, the effects of formulation variables on the defined crit-
ical parameters are examined to optimize protein stability. Which variables are
most important depends of the formulation type chosen. For example, resistance
to agitation and/or accidental freezing is a critical property for an aqueous for-
mulation, which would not be a concern for a lyophilized formulation. Similarly,
choosing excipients that provide a glassy matrix to stabilize the protein is only a
concern for dried formulations.

Formulatiou Optious 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 distribu-
tion, and market competitiveness. Having a clear plan about what type of for-
mulation is desired will allow one to design better formulation studies. Liquid
formulations have been generally preferred due to the convenience of manufac-
turing and use. However, protein drugs may not be stable enough to be handled
as a liquid formulation. Dried formulations (e.g., lyophilized) or suspension for-
mulations (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. As details of the stability issues are discussed in other chapters in this
book, only practical issues regarding these different types of formulations will be
summarized here.
Liquid Formulations. It is important to understand that developing condi-
tions to keep proteins stable in a liquid form for a pharmaceutically relevant
Practical Approaches to Protein Formulation Development 11

storage time (e.g., two years) is not a simple task. For most proteins, including
relatively stable ones, at least some degradation should be expected even during
refrigerated storage. Unless there is strong evidence supporting that the protein
remains stable for two years and the evidence is supported by a broad spectrum
of analytical methods, one needs to be very careful about the final decision to
market a liquid formulation. Physical or chemical changes that have low ac-
tivation energy should be monitored especially carefully under real storage
conditions. If there is a degradation product that can significantly affect the phar-
maceutical quality at its minimum concentrations (e.g., formation of particulates)
great care must be taken to assure that such a product does reach unacceptable
levels during real-time storage. If long-term storage studies are not implemented
until late in the product development process, there is a risk that problems due
to unacceptable levels of degradation products will not be discovered in time to
test alternative formulations. In addition to stability during storage, temporary
exposure to temperatures outside the recommended conditions should also be
tested because it can affect the quality of the drug. Also, results obtained from
multiple freeze-thawing cycles will be useful to determine if the formulation can
accommodate unexpected freezing during distribution and storage. Likewise, sen-
sitivity to agitation or surface denaturation needs to be understood to support for-
mulation choices as well as shipping and handling guidelines. For formulations
containing low protein concentration, special attention is required to avoid impu-
rity-related stability issues (e.g., oxidation fostered by metal contaminants in
excipients) .
Solid Dosage Forms. Recent improvements in devices designed for easier
use of lyophilized products, e.g., dual chamber syringes, dual chamber cartridges
and convenient reconstitution devices, have helped pharmaceutical industries to
develop lyophilized products without too many concerns surrounding patient
compliance issues. Because lyophilized products have less stability-related issues
and have a much greater potential tolerance for room-temperature storage, many
biopharmaceutical industries consider the lyophilized formulation as a default
option. In fact, lyophilization has been widely utilized to overcome various sta-
bility issues of labile proteins. However, it is important to note that lyophilization
can present its own challenges, particularly in designing appropriate formulations
and economic cycles. Detailed discussion about the rational design of stable
lyophilized formulations is available in another chapter in this book.
In addition to the required stability, a successful lyophilized formulation
should also have the desired physical properties of the dried powder, e.g., rugged-
ness of the cake and the maintenance of the physical states for each of the
ingredients. For example, crystallization of an initially amorphous excipient
during storage can lead to unacceptable degradation of the protein product and loss
of cake structure. Furthermore, optimal water content for protein stability should
be studied and controlled during storage. It is preferable to define an acceptable
12 Byeong S. Chang and Susan Hershenson

range of water contents to provide flexibility in the manufacturing process. To

assure that the appropriate water content is maintain, the integrity of container/
packaging should be monitored in a high relative humidity environment.
The identity and volume of the reconstitution medium should also be clearly
defined, because the use of injectable fluids other than the recommended one may
compromise the quality of the product (e.g., solution tonicity or protein stabil-
ity). Reconstitution should be convenient and rapid, and the stability of the recon-
stituted solution during handling and delivery needs to be demonstrated.
Only recently have· spray-dried formulations become commonly used with
the advent of novel protein delivery technologies. For example, spray-dried
powders have been useful for developing products that require uniform particle
size (e.g., pulmonary inhalation for systemic delivery). Similar issues to the
lyophilized formulation apply to the spray-dried formulation, and the details of
development of spray-dried formulations are presented in another chapter in this
book. In addition to protein stability, physical properties pertinent to powder pro-
cessing and powder stability (e.g., flowablity, hygroscopicity, agglomeration,
density and crystallization of excipients) need to be well characterized.
Single Dose and Multidose Forms. Most protein pharmaceuticals are mar-
keted as single dose forms. However, multidose formulations are useful when the
dose needs to be split (e.g., dose titration or dose combination). Multidose for-
mulations are distinguished by the presence of preservatives in the formulation,
which prevent microbial contamination and/or growth during multiple disruptions
of container closure integrity. In general, the addition of preservative(s), regard-
less of the preservative used, significantly changes the stability profiles of
proteins (Maa and Hsu, 1996; Fransson et aI., 1997; Lam et aI., 1997). In some
extreme cases, visible precipitation and aggregation have been reported. There-
fore, the effect of various preservatives on the stability of protein should be
carefully examined. Other experiments required to qualify a multidose for-
mulations include the preservative effectiveness test and the stopper self-sealing
test. Detailed procedures and specification are available from the USP (U.S.
Pharmacopeia), the Ph. Eur. (European Pharmacopeia) and the IP (Japan
Pharmacopeia) .
Desired properties of the ideal preservative include: effectiveness at low
concentration against a wide variety of organisms; chemical stability; solubility;
compatibility with the protein drug, excipients and auxiliary agents; free from
objectionable odor, taste, color and stinging; and non-toxic and non-sensitizing
both internally and externally at the required concentration. Also, it must not
absorb, penetrate, or interact with containers or closures (Thompson, 1998). For
further details on preservatives, readers are referred to Regulatory Document 4
in the Appendix and Thompson (1988). Examples of typical preservatives used
for parenteral protein pharmaceuticals are benzyl alcohol, phenol, m-cresol and
benzalkonium chloride.
Practical Approaches to Protein Formulation Development 13

Typical Protein Stability Problems: Causes and Solutions

Table 6 summarizes typical stability problems observed during protein for-

mulation development and potential methods to solve each problem. The list does
not represent the complexity of multiple problems that can be experienced with
a given protein. Formulation research should be designed to handle each protein
based on its unique stability profile.

Optimization of Formulation Variables

Overview of the Process. The optimization of formulation variables for

product stability is the most critical part of protein formulation development.
Various formulation excipients and buffers (Table 7) can be utilized and must,
therefore, be chosen to maximize the pharmaceutical quality of the product (i.e.,
stability and activity) without introducing significant side effects. Among the
listed formulation variables, the most powerful one is pH. Problems associated
with the physical properties of a protein, e.g., precipitation due to solubility and/or
stability, are generally very difficult to manage by other formulation means.
Optimization of pH is a simple but very useful solution for such problems (Kol-
venbach et aI., 1997). Most chemical reactions also are affected by pH, e.g.,

Table 6.
Typical Stability Problems Observed in Protein Pharmaceuticals
Problems Potential causes Possible solutions

Non-covalent aggregation Solubility, structural changes, pH, ionic additives, amino

heat, shear, surface, acids, surfactants, protein
denaturants, impurities concentration, raw
material purity
Covalent aggregation Disulfide scrambling, other pH, inhibit non-covalent
unknown 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 Excipient purity, free-radical
radicals, metals, light, impurity scavenger, active oxygen
scavengers
Surface denaturation, Low protein concentration, Surfactants, protein
adsorption specific affinity, protein concentration, pH
hydrophobicity
14 Byeong S. Chang and Susan Hershenson

Table 7.
Important Components of Protein Formulations
Fonnulation
Variables Desired attributes Examples
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 Salts, amino acids, surfactants
low concentrations
Buffer Good buffering capacity, stable to Phosphate, acetate, histidine,
temperature change, stable to glutamate
freezing, good safety record
Tonicity modifier; Inert, good safety record Sodium chloride, sorbitol,
bulking agent mannitol, glycine

deamidation, cyclic imide formation, disulfide scrambiing, peptide bond cleav-

age, and oxidation (see reviews listed above). Other functional excipients should
be also carefully evaluated for the benefit of the product (e.g., use of sucrose to
stabilize protein during lyophilization and storage in the dried solid).
In addition to their intended use, excipient candidates should be also quali-
fied as appropriate pharmaceutical ingredients. Generally, it is preferred to select
excipients that have been used in marketed products with a relevant route of
delivery. Further, it is preferable if these excipients have been used with similar
frequency of dosing, history of chronic use and similar patient populations.
Otherwise, the approval and safety of the excipients need to be carefully
examined. If the excipient is considered safe with a solid scientific basis or has
a proven clinical safety record, it can be considered equivalent to approved ex-
cipients. The list of excipients used for parenteral pharmaceuticals is available in
the literature (Powell et aI., 1998; Nema et aI., 1997; Appendix Regulatory Doc-
ument 5). When it is necessary to introduce other excipients with minimum safety
records, a significant risk associated with the excipients will be added to the
product development and additional pre-clinical and clinical studies may be
needed.
Another important requirement in qualifying an excipient is the purity of the
raw material. Depending on the historical use as a pharmaceutical ingredient,
several different pharmaceutical grades are available, e.g., USP (U.S. Pharma-
copeia), Ph. Eur. (European Pharmacopeia) and IP (Japan Pharmacopeia). These
pharmaceutical grade materials should be considered as a primary resource, but
the quality provided may not be good enough for specific product development.
For example, significant stability problems can be found with some impurities
Practical Approaches to Protein Formulation Development 15

even at concentrations below their specification, e.g., metal ions, peroxides, pro-
teases and reducing sugars. These problems are more prominent in low protein
concentration products due to a high impurity-protein ratio, although problems
like visible precipitation of the protein may be independent of protein concen-
tration. If adjustment in the existing specification is necessary, it is critical to look
into the availability of GMP quality raw materials with modified specifications
as early as possible, once the potential problem is identified.
The use of excipients derived from animals (e.g., Tweens) or humans (e.g.,
human serum albumin) should be avoided if possible due to the risk associated
with transmissible diseases like bovine spongiform encephalopathy, Creutzfeldt-
Jakob Disease, hepatitis virus and HIY. Numerous regulatory guidelines have
been issued to discourage the use of animallhuman-derived excipients (Appen-
dix Regulatory Documents 6,7,8). When animal-derived excipients have to be
included in the product, the manufacturer will need to demonstrate that their
selection is fully justified.
Design of the Study. A design for the formulation optimization study should
be in place before commencing the work. A typical study protocol will include
the following information:
- 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
The study can be more efficiently designed by utilizing experimental design
software packages, which will help to minimize the resources required for sample
preparation, analyses, and data analysis.

Necessary Studies for Formulation Development

Storage Stability Study. Documenting that the formulation will keep the
protein stable until the desired expiry can be the most time-consuming part of
16 Byeong S. Chang and Susan Hershenson

formulation development. The expiry requirements are determined by distribu-

tion not regulatory requirements. One year is probably too short for effective
manufacturing and distribution through normal channels. In general, a shelf life
of 18 month is considered acceptable for commercialization. Results obtained
from accelerated stability studies are useful for predicting potential degradation
products and appropriate analytical methods, but whatever the actual shelf life,
it must be supported by sufficient real-time storage data to obtain regulatory
approval. Thus, it is important to establish a final formulation and start the real-
time storage studies as early as possible during product development.
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. For example, mixing solutions in a large stainless
steel tank, pumping solutions through stainless steel tubing, filtration and filling
through a high-speed filling machine can introduce unexpected stresses to the
protein. An increase in the formation of particulates, along with a loss of proteins
due to surface adsorption and aggregation, has routinely been observed. It is
important to expose the formulation to equivalent stresses and make sure that
no formulation adjustment is necessary to accommodate the manufacturing
processes. Again, this testing should be done as early in the development process
as possible.
Transportation, Handling and Delivery Study. Unexpected environmen-
tal changes can be encountered during the distribution and handling of products
(e.g., accidental freezing, exposure to temperatures different from the recom-
mended conditions, vigorous agitation, etc). It is critical to develop the formula-
tion with these stresses in mind because they can compromise the quality of the
product.
Also, during administration to the patient, proteins can be exposed to dif-
ferent types of stresses introduced by the device and the routes of delivery. Exam-
ples include incompatibility with the delivery device (e.g., protein aggregation
induced by exposure to tubing surfaces) and/or concomitant medication (e.g.,
protein aggregation induced by co-administered antibiotics).
Preclinical and Clinical Studies. Results documenting the maintenance of
the biophysical and biochemical properties of the protein are essential for a final
formulation decision. Before finalizing the formulation, it is also important to
confirm that it does not affect critical in vivo biological properties of the protein
(e.g., activity, pharmacokinetic profile, and toxicity profile). Maintenance of a
protein's biophysical properties can be examined using various structural analy-
ses (e.g., circular dichroism, fluorescence and infrared spectroscopies, etc.). It is
possible to determine the biochemical equivalence of the protein pharmaceutical
by in vitro activity and/or preclinical in vivo bioassays. Results supporting the
toxicity profile of the formulation can be generated by both preclinical and
clinical studies.
Practical Approaches to Protein Formulation Development 17

Strategies to Overcome Difficult Formulation Problems

Occasionally, problems are encountered that are difficult to overcome with

conventional formulation approaches. It is possible to conduct additional studies
to confirm that the problem is pharmaceutically acceptable. On the other hand,
unique formulation approaches can be introduced to address specific problems.
Examples of this approach are provided below.
Qualification of Degradation Products. If decreasing the amount of degra-
dation product below the specified threshold is not feasible, then the degradation
product can be defined as a drug substance by demonstrating that it does not affect
the pharmaceutical quality of the drug product, i.e., safety and efficacy (Appen-
dix, Regulatory Document 1). If there is not sufficient information available,
additional qualification studies recommended in the regulatory guidance can be
carried out (see the discussion above). The experience of having these degrada-
tion products in the materials used for clinical studies can sometimes be very
useful because the results obtained can provide useful insight into the clinical
implication of the degradation products.
Site-directed Mutagenesis to Improve Properties. Some problems related
to the intrinsic properties of a protein cannot be overcome unless a change in
the sequence is introduced. After careful research is carried out to identify the
problematic region or residue, the protein can be engineered for better physical
and/or chemical properties. For serious problems like precipitation due to
insolubility or poor stability, changes in protein sequence have been proven effec-
tive for improving physical properties (Murby et aI., 1995; Roig and Kennedy,
1995).
Chemical Modifications. The physical properties of proteins can be
improved by modifying problematic amino acid side chains with small com-
pounds or large polymers (e.g., attachment of polyethylene glycol) (Fagain,
1995; Guerra et aI., 1998; Francis et aI., 1998). In addition, there are other
desirable improvements that can be achieved by the conjugation chemistry
(e.g., enhanced pharmacokinetic profiles, reduced immunogenecity, enhanced
adsorption, etc.).
Unconventional Dosage Forms. Some protein stability problems can be
resolved by introducing novel approaches in the formulation. Examples include
suspension formulations (Defelippis et aI., 1998), microencapsulation with
cyclodextrin (Brewster et aI., 1991), suspension of dry-powder in non-aqueous
vehicle (Knepp et aI., 1998), and use of non-aqueous vehicles with hydrophobic
ion-pairing (Manning et aI., 1995).
18 Byeong S. Chang and Susan Hershenson

FORMULATION IN COMMERCIAL PRODUCT DEVELOPMENT

Critical Formulation Decisions During Pharmaceutical Development

A brief summary of the important phases of a commercial product develop-

ment is shown in Figure 1. Ideally, a stable formulation would be available during
the early discovery research period to ensure that the protein is in its active and
stable form during critical feasibility studies. However, development of a stable
formulation for each drug candidate is not practically possible because such an
approach would take too much time and too many resources. Generally, formu-
lation development starts after the decision is made to start clinical trials. A

Preliminary
formulation

.-----
development
Preclinical Research
Process Development
GLP/GMP stability Commercial
Initial formulation
development
formulation(s)

Official stability
Phase "" clinical study start for
commercial
Phase III clinical
.----- formulation

License
Commercial Second
application
generation
formulation formulation(s)

.-----
development
Commercial
product

Figure 1. Diagram showing commercial formulation development process.

Practical Approaches to Protein Formulation Development 19

reliable formulation is required to support various preclinical and clinical studies.

The formulation can be further improved later to satisfy the clinical, marketing,
and regulatory needs. Although formulation development does not necessarily
appear rate-limiting during this improvement period, it is important to understand
that changing a formulation may require much more supporting work, including
additional clinical trials, which can take years.

Formulation for Early Preclinical and Clinical Studies

When a promising protein drug candidate is identified from preliminary fea-

sibility studies, a decision will be made to introduce the drug to clinical studies. In
order to obtain regulatory approval to initiate the clinical trials, it is required to
demonstrate safety, manufacturing capability, stability of the drug and repro-
ducibility in studying the drug. A sufficiently stable formulation is necessary at this
stage because it is important to maintain the quantity and quality of the protein for
all research studies from which results will be used for registration of the product
with regulatory agencies. Therefore, formulation development becomes a rate-
limiting process at this early stage. Formulations can be stored frozen or
lyophilized for this purpose. At this stage in product development, the shelf-life
requirement is determined by the logistics of supplying drug for clinical trials.

Commercial Formnlation

Commercially viable and market competItIve formulations have some

common features. Most of all, the formulation should maintain the safety and
efficacy profile of the protein drug during all the handling and uses specified on
the label. Since commercial distribution channels are not equipped for frozen
products, shipping and storage at refrigerated temperature or higher are required.
Sufficient shelf life needs to be determined under conditions to which the product
will be exposed in the commercial distribution system. Various systematic studies
need to be carried out to comply with regulatory requirements for registration. It
takes 1-2 years to collect all of these results, so the commercial formulation is
developed while preclinical studies or early clinical trials (with the preliminary
formulation) are in progress. If possible, the commercial formulation should be
introduced before the pivotal clinical trial because clinical experience is the most
effective way to confirm the safety and efficacy aspects of the formulation. In
addition, formulation changes after this point may introduce formidable chal-
lenges to the clinical program and to obtaining regulatory approval.
20 Byeong S. Chang and Susan Hershenson

Regulatory Issues in Formulation Development

Incorporating regulatory guidelines into the formulation development is not

only useful to develop high quality pharmaceutical formulations for maximum
benefit to the patients, but also critical to prepare complete documents necessary
for regulatory approval for commercialization of the drug. Readers who wish to
obtain a comprehensive understanding of the guidelines are referred to the rele-
vant documents published by regulatory agencies. In this section, a brief summary
of the guidelines related to the formulation issues is provided.
Guidelines for Stability Studies. Comprehensive guidelines can be
obtained from regulatory agencies (Appendix, Regulatory Documents 9-12) and
review articles in the literature (Grimm, 1998; Kommanaboyina and Rhodes,
1999; Matthews, 1999). These guidelines generally cover the formal stability
studies for bulk drug material, in-process samples and final formulated product.
Although some issues may not be relevant for the earlier formulation develop-
ment work, these guidelines provide key elements for designing stability studies.
The guidelines provide information such as what types of experiments should be
included in the study protocol, how to propose a stability-indicating profile, and
what analytical results are needed to define the purity of the product and the mol-
ecular characteristics of degradation products. Another important piece of infor-
mation provided in the guidelines is the definition of proper experimental
conditions, e.g., definition of storage temperature, humidity, light strength, and
accelerated and stressed conditions. The guidelines also provide the official pro-
tocols to evaluate the results obtained from stability studies.
Results Required to Apply for Regulatory License for a Drug Product.
Detailed information regarding the formulation is presented at the Development
Pharmaceutics section in the CMC section of regulatory applications (Appendix,
Regulatory Docoments 13-16). All of the essential information required to apply
for regulatory approval of protein formulations can be found in Note for Guid-
ance on Development Pharmaceutics (Appendix Regulatory Document 17) and
Development of Pharmaceutics for Biotechnological and Biological Products
(Appendix, Regulatory Document 8). It is important to note that this is currently
a European requirement although the international harmonization process may
eventually introduce it to U.S. regulatory requirements. A brief summary of the
important formulation information is shown in Table 8.
Results Required to File Formulation Amendments. A formulation
change can introduce substantial potential adverse effects on the identity,
strength, quality, purity or potency of the product as related to the safety or effec-
tiveness of the product. For this reason, it is required that a supplement to the
approved license application be submitted. This supplement should include a
detailed description of the proposed change. It should also include methods and
results for the studies performed to evaluate the effect of the change on the
Practical Approaches to Protein Formulation Development 21

Table 8.
Summary of Information Included in Regulatory Applications
General information Comments specific to proteins

Active substance Compatibility with excipients and other Structural elements responsible
(protein) combined products, and for the biological activity;
physicochemical characteristics good coverage of degradations
occurring during storage,
manufacturing, and delivery.
Excipients Function of each excipients, justification
for their inclusion, and compatibility
with other excipients, choice of quality
Formulated Overage, physicochemical parameters, Stability of structure in terms of
products components with appropriate results biological activity; formulation
supporting intended purpose; optimization for both
compatibility with diluent, device, and manufacturing process and
other drugs in contact before delivery; stability
critical physical properties
Packaging Integrity of container and closure during Adsorption, denaturation at the
material storage, reconstitution, admixture, interface, and aggregation of
dilution; sorption to container, leaching, the surface-denatured protein;
and dose reproducibility reconstitution of dry-powder
formulation
Manufacturing Manufacturing process for the preparation Results to support the quality
process of formulation and its justification, and stability of the protein
appropriate method of sterilization and during the manufacturing
justification process; membrane filtration
under aseptic conditions
sufficient

product's identity, strength, quality, purity, and potency of the product, as related
to the product's safety or effectiveness (Appendix, Regulatory Documents
18-20). When the degradation profile is changed qualitatively or quantitatively,
it is recommended to follow the impurity-related guidelines discussed above
(Appendix, Regulatory Documents 2,3). The manufacturer must obtain approval
of the supplement by FDA prior to distribution of the product made using the
change. In general, the following studies (Table 9) can be carried out to confirm

Table 9.
Studies Needed to Support Change in Formulation
Research requirements Supporting results
Purity Stability, compatibility, Structural analyses
Potency In vitro/in vivo bioassays, preclinical and/or clinical pharmacokinetic
comparability, clinical efficacy
Safety Preclinical safety, clinical safety
22 Byeong S. Chang and Susan Hershenson

that the change in the formulation will not affect the safety and effectiveness of
the product. It is recommended to consult the regulatory representative to find
out how much additional information may be needed for the approval of the
changed formulation.

APPENDIX: LIST OF REGULATORY DOCUMENTS

1. International Conference on Harmonization: Guidance on specifications: Test

procedures and acceptance criteria for Biotechnological/Biological Products.
Federal Register: August 18, Volume 64, Number 159, pp. 44928-44935
(1999).
2. International Conference on Harmonization: Impurities in New Drug Prod-
ucts. FDA Q3B (11/97).
3. Guidance for Industry. ANDAs: Impurities in Drug Products. Draft guidance.
FDA (12/98).
4. Note for Guidance on Inclusion of Antioxidants and Antimicrobial Preser-
vatives in Medicinal Products (CPMP/CVMP/QWP/115/95) EMEA (7/97).
5. Inactive ingredient guide: inactive ingredients for currently marketed drug
products. (1996) FOI Services, Inc. Rockville, MD.
6. Note for guidance on minimizing the risk of transmitting animal spongiform
encephalopathy agents via medicinal products. EMEA (4/99).
7. Note for guidance on plasma-derived medicinal products. EMEA (7/98).
8. Development Pharmaceutics for Biotechnological and Biological Products.
Annex to Note for Guidance on Development Pharmaceutics (CPMPI
QWP/155/96). EMEA (10/1999).
9. International Conference on Harmonization: Stability testing of new drug
substances and products. Federal Register, Sept. 22, Volume 59, Number 183,
pp. 48754-48759 (1994).
10. International Conference on Harmonization: Final guideline on stability
testing of biotechnological/biological products. Federal Register, July 10,
Volume 61, Number133, pp. 36466-36469 (1996).
11. Guideline for Industry: Stability testing for drug substances and drug prod-
ucts: draft guidances. FDA, (6/98).
12. International Conference on Harmonization: Guidelines for the photostabil-
ity testing of new drug substances and products. Federal Register, May 16,
Volume 62, Number. 95, pp. 27115-2712 (1997).
13. Guideline for Submitting Documentation for the Stability of Human Drugs
and Biologics FDA. (2/87).
14. Guidance for Industry. For the Submission of Chemistry, Manufacturing and
Controls and Establishment Description Information for Human Plasma-
Practical Approaches to Protein Formulation Development 23

Derived Biological Products, Animal Plasma or Serum-Derived Products.

FDA (2/99).
15. Guidance for Industry. INDs for Phase 2 and 3 Studies of Drugs, Including
Specified Therapeutic Biotechnology-Derived Products Chemistry, Manu-
facturing, and Controls Content and Format (Draft guidance) FDA (2/99).
16. Content and Format of Investigational New Drug Applications (INDs) for
Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic,
Biotechnology-Derived Products, FDA (11/95).
17. Note for Guidance on Development of Pharmaceutics (CPMPIBWP/328/99).
EMEA (0111998).
18. Demonstration of Comparability of Human Biological Products, Including
Therapeutic Biotechnology-Derived Products, April, 1996.
19. Guidance for Industry: Changes to an Approved Application. For Specified
Biotechnology and Specified Synthetic Biological Products, 21 CFR 601.12,
314.70; July 24, 1997, Vol 62. No. 142.
20. Guidance for Industry: Changes to an Approved Application: Specified
Biotechnology and Specified Synthetic Biological Products; July 1997.

REFERENCES

Ahem, T.J. and Manning, M.C., 1992a. Stability of protein pharmaceuticals, Part A: Chem-
ical and physical pathways of protein degradation. Pharm. Biotech. Ser. Volume 2.
Plenum Press, N.Y.
Ahem, T.J. and Manning, M.e., 1992b. Stability of protein pharmaceuticals, Part B: In
vivo pathways of degradation and strategies for protein stabilization. Pharm. Biotech.
Ser. Volume 3. Plenum Press, N.Y.
Arakawa, T., Prestrelski, S., Kinney, W., and Carpenter, J.P', 1993. Factors affecting short-
term and long-term stabilities of proteins. Adv. Drug Delivery Rev. 10: 1.
Brewster, M.E., Hora, M.S., Simpkins, J.w., and Bodor, N., 1991. Use of 2-
hydroxypropyl-beta-cyclodextrin as a solubilizing and stabilizing excipient for
protein drugs. Pharm. Res. 8:792.
Cleland, J.L., Powell, M.P., and Shire, S.J., 1993. The development of stable protein
formulations-A close look at protein aggregation, dearnidation and oxidation. Crit.
Rev. Ther. Drug 11 :60.
Chen, T., 1992. Formulation concerns of protein drugs. Drug Dev. Ind. Pharmacy, 18:1311.
Defelippis, M.R., Bakaysa, D.L., Bell, M.A., Heady, M.A., Li, S., Pye, S., Youngman,
K.M., Radzuik, J., and Frank, B.H., 1998. Preparation and characterization of a
cocrystalline suspension of [Lys(B28),Pro(B29)] human insulin analogue. J. Pharm.
Sci. 87:170.
Fagain, e.O., 1995. Understanding and increasing protein stability. Biochimica et Bio-
physica Acta. 1252: 1.
24 Byeong S. Chang and Susan Hershenson

Francis, G.B., Fisher, D., Delgado, C., Malik, E, Gardiner, A, and Neale, D., 1998. PEGy-
lation of cytokines and other therapeutic proteins and peptides: the importance of bio-
logical optimisation of coupling techniques Int. J. Hematology. 68:1.
Fransson, J., Hallen, D., and Florin-Robertsson, E., 1997. Solvent effects on the solubil-
ity and physical stability of human Insulin-like Growth Factor I. Pharm. Res. 14:606.
Grimm, W., 1998. Extension of the international conference on harmonization tripatic
guieline for stability testing of new drug substances and products to countries of cli-
matic zones III and IV. Drug Dev. Indust. Pharm. 24:313.
Guerra, P.I., Acklin, C., Kosky, AA, Davis, J.M., Treuheit, MJ., and Brems, D.N., 1998.
PEGylation prevents the N-terminal degradation of megakaryocyte growth and devel-
opment factor Pharm. Res. 15:1822.
Herron, IN., Jiskoot, w., and Crommelin, D.J.A, 1995. Physical methods to characterize
pharmaceutical proteins. Pharm. Biotech. Ser. Volume 7. Plenum Press, N.Y.
Jones, AJ.S., 1993. Analysis of polypeptides and proteins Adv. Drug Del. Rev.
10:29.
Knepp, V.M., Muchnik, A., Oldmark, S., and Kalashnikova, L., 1998. Stability of non-
aqueous suspension formulations of plasma derived factor IX and recombinant human
alpha interferon at elevated temperatures. Pharm. Res. 15:1090.
Kolvenbach, C.G., Narhi, L.O., Philo, J.S., Li, T., Zhang, M., and Arakawa, T., 1997. Gran-
ulocyte-colony stimulating factor maintains a thermally stable, compact, partially
folded structure at pH 2 J. Pept. Res. 50:310.
Kommanaboyina, B. and Rhodes, c.T., 1999. Trends in stability testing with emphasis on
stability during distribution and storage. Drug Dev. Indust. Pharm. 25:857.
Lam, X.M., Patapoff, T.w., and Nguyen, T.H., 1997. The effect of benzyl alcohol on
recombinant human interferon-gamma Pharm. Res. 14:725.
Maa,Y.E and Hsu, C.C., 1996. Aggregation of recombinant human growth hormone
induced by phenolic compounds Int. J. Pharm. 140:155.
Manning, M.C., Matsuura, J.E., Kendrick, B.S., Meyer, J.D., Dorrnish, J.J., Vrkljan, M.,
Ruth, J.R, Carpenter, J.E, and Shefter, B., 1995. Approaches for increasing the solu-
tion stability of proteins Biotech. Bioeng. 48:506.
Manning, M.C., Patel, K., and Borchardt, RT., 1989. Stability of protein pharmaceuticals.
Pharm. Res. 6:903.
Matthews, B.R, 1999. Regulatory aspects of stability testing in Europe. Drug Dev. Indust.
Pharm. 25:831.
Murby, M., Samuelsson, E., Nguyen, T.N., Mignard, L., Power, D., Binz, H., Dhlen, M.,
and Stahl, S., 1995. Hydrophobility engineering to increase solubility and stability of
a recombinant protein from respiratory syncytial virus. Eur. J. Biochem. 230:38.
Nema, S., Washkuhn, R.J., and Brendel, R.J., 1997. Excipients and their use in injectable
products PDA J. Pharm. Sci. Technol. 51:166.
Note for Guidance on Inclusion of Antioxidants and Antimicrobial Preservatives in Med-
icinal Products, 1997. (CPMP/CVMP/QWPI115/95) EMEA (7/97).
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.
Powell, M.E, Nguyen, T., and Ba10ian, L., 1998. Compendium of excipients for parenteral
formulations. PDA J. Pharm. Sci. Technol. 52:238.
Practical Approaches to Protein Formulation Development 25

Reubsaet, J.L.E., Beijnen, J.H., Bult, A, Van-Maanen, R.J., Marchal, J.AD., and Under-
berg, W.J.M., 1998. Analytical techniques used to study the degradation of proteins
and peptides: chemical instability 1. Pharm. Biomed. Anal. 17:955.
Roig, M.G. and Kennedy, J.F., 1995. Perspectives for biophysicochemical modifications
of enzymes. 1. Biomaterials Sci. Polymer Ed. 7:1.
Thompson, J.E., 1998. Practical Guide to Contemporary Pharmacy Practice. Lippincott
Williams & Wilkins, Hagerstown, MD.
Volkin, D.B., Mach, H., and Middaugh, C.R., 1997. Degradative covalent reactions impor-
tant to protein Stability. Malec. Biotech. 8:5.
Wang, Y.J. and Hanson, M.A, 1988. Parenteral formulations of proteins and peptides: sta-
bility and stabilizers. 1. Parent. Sci. Technol. 42:SS4.
Wang, Y.J. and Pearlman, R., 1993. Stability and characterization of protein and peptide
drugs: case histories. Pharm. Biotech. Ser. Volume 5. Plenum Press, N.Y.