ICH Q5 GUIDELINES

Stability Testing of Biological/Biotechnological Products

 Introduction

• The ICH Q5C guideline was released in 1995 to supplement guideline Q1A(R2) by
specifically addressing the unique characteristics of biological drugs compared with
small molecule drugs.
• The document covers a wide range of well‐characterized proteins and polypeptides,
either purified from natural sources or manufactured from recombinant DNA
technology. The guidance also applies to both conventional and recombinant vaccines.
• Q5C makes it very clear that for biologicals, “long-term, real-time, real-condition”
stability studies are required to establish the requested storage period.
 Purposes of stability studies :

• The studies and the storage conditions evaluated serve multiple purposes at different
stages of product development and the product life cycle.
Analytical method Development :
• At the very early stages of product development, a suite of analytical methods is
developed to facilitate initial process and formulation development.
• A subset of analytical methods should be stability-indicating to ensure that all
degradants can be monitored and controlled during manufacturing and storage.
Formulation Development :
• One widely adopted approach of formulation development is to subject the product in
different formulations to various stability/stress conditions.

Degradation Pathway Elucidation :

• Stability studies, especially accelerated and stress stability studies, can be used to
elucidate degradation pathways for the product being evaluated.
• Degradants that appear during recommended storage and accelerated conditions
should be characterized and their criticality should be evaluated.
• Stress studies are used to generate higher amounts of these degradants inorder to
facilitate the elucidation of their identity (structure).
Expiry (Use-by-date) Establishment :

• The shelf life of drug substance and final drug product should be established primarily
based on “long-term, real-time, real-condition” stability studies.
• Any unexpected, out-of-trend, or out-of-specification results should be adequately
investigated following the established internal quality systems of the testing
organization and of the sponsor of the study.

Storage/Transportation/In-Use Condition Excursions:

• During manufacturing, handling, transportation, and in-use in clinics, drug substance or
drug product may sometimes be subjected for short periods of time to conditions that
are outside of the recommended storage conditions
• Stability studies conducted under accelerated conditions, including higher than optimal
temperature and relative humidity (as appropriate) as well as multiple freeze–thaw
cycles, can provide the data needed to justify the acceptability of drug substance or
drug product subjected to an occasional excursion in storage.

Comparability/Similarity:
• Stability studies are an indispensable component of a comparability exercise.
• The assumption is that any degradation pattern or rate difference observed could
represent a difference in the products under consideration that may or may not be
detected through other comparability exercises.
• For products such as monoclonal antibodies, typical accelerated (e.g., 25°C) and stress
conditions (37 or 40°C) may not be harsh enough to degrade the products. In this case,
harsher forced degradation conditions (e.g., 60 or 10°C below the product melting
temperature) might be used.
• Since the goal of such degradation studies is to demonstrate comparability and these
data are not used to establish or extrapolate expiry, different storage and containers
than the ones used in typical stability studies may be used.
• For example, to establish the shelf life for a lyophilized drug product, the stability
study must be done using product in the lyophilized state stored in the
actual/representative drug product containers.
• However, to demonstrate comparability before and after a process change, the
lyophilized drugs can be reconstituted in a buffer and transferred from the original
containers (e.g., vials) into different containers to facilitate quick comparison of the
degradation profiles and rates under the same conditions for the two comparator
products.
 Degradation Pathways of Therapeutic Proteins:

• Proteins undergo various degradation pathways during manufacturing, storage,

transportation, and handling by end users.
• The most commonly observed protein degradation pathways are aggregation,
fragmentation, oxidation, deamidation/isomerization, disulfide bond
scrambling/exchange, glycation and diasialylation.
Aggregation:
• Aggregation results when a monomer molecule associates with other monomers
through covalent or noncovalent interactions to form dimers, trimers, multimers, or
high molecular weight species.
• Aggregation results when a monomer molecule associates with other monomers
through covalent or noncovalent interactions to form dimers, trimers, multimers, or
high molecular weight species.
• For therapeutic proteins, both aggregates and particles pose a safety concern due to
their potential immunogenicity.
• Protein aggregation occurs due to various causes and under various circumstances,
such as incorrect folding during protein expression, perturbation of the native
conformation during purification, formulation, freeze–thawing, freeze drying,
ultrafiltration/diafiltration, vial or syringe filling, pumping, transportation, or
storage.
• The analytical methods that are most commonly used to detect protein aggregation
are SEC, analytical ultracentrifugation (AUC), dynamic or static light scattering,
and asymmetric flow field flow fractionation
 Fragmentation:
• Fragmentation refers to the disruption of peptide (amide) bonds within a protein as a result of
chemical or enzymatic reactions.
• Heat, certain pH, free radicals, trace metals, and light can all facilitate protein fragmentation.
Peptide bonds between certain amino acids (e.g., those between aspartic acid and proline
residues) are also points of potential degradation, being inherently susceptible to chemical
hydrolysis.
• Residual proteases (e.g., cathepsin family) that have been reported to exist in many biologics
can result in fragmentation of the biologic under conditions favoring protease activity.
• Commonly used methods for detecting and quantifying fragments include SEC either under
native or denatured conditions, reduced and nonreduced sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and the capillary versions (CE-SDS) and
reversed-phase high performance liquid chromatography (RP-HPLC).
Oxidation:

• Heat, light, trace metals, and oxygen radicals can each oxidize susceptible amino acids,
especially methionine, tryptophan, and histidine.
• Methionine is the amino acid residue most susceptible to oxidation.
• Tryptophan oxidation is also commonly observed during stability studies, especially in
photostability studies.
• Polysorbate used in protein formulations can degrade in the presence of light or metals
and can induce subsequent protein oxidation.
• Oxidation of a biologic can be monitored by RP-HPLC peptide mapping with or
without mass spectrometry (MS).
• Methionine oxidation in the Fc region of a monoclonal antibody can also be
measured by protein A affinity chromatography.
• Protein oxidation has been reported to affect a variety of protein properties, including
binding affinity to antigen, effector functions, and serum half life. Because of this,
oxidation is considered one of the major concerns in therapeutic monoclonal
antibody development.

Deamidation/Isomerization:
• Protein deamidation refers to the loss of an amide functional group from
asparagine(Asn) or glutamine (Gln) residues.
• Subsequent hydrolysis of the succinimide intermediate yields a hydroxyl group,
forming aspartic acid (and isoaspartic acid) or glutamic acid residues.
• In addition to the susceptibility due to the protein’s intrinsic structure previously
mentioned, the deamidation rate depends on pH, temperature, ionic strength, and
buffer type.
• Deamidation is typically favored at high pH, while isomerization can happen under
acidic conditions.
• The impact of deamidation on the biological activity of the therapeutic protein is
highly dependent on the location of the modification.
• For example, deamidation/isomerization in the CDR of monoclonal antibodies has
been reported to impact potency, while deamidation in the Fc region of the
monoclonal antibody molecule is generally considered to have much less impact
Disulfide Bond Scrambling/Exchange:

• Disulfide bonds play a critical role in maintaining the structural integrity of therapeutic
proteins.
• Some cysteines in the protein backbone may exist in the reduced form in which a free
thiol group is available for reaction. Such free thiols may induce disulfide bond
scrambling and induce protein aggregation.
Glycation:
• Glycation occurs when a protein is incubated in the presence of a reducing sugar, such
as fructose or glucose.
• The amine group of an amino acid, typically lysine (Lys), reacts with the carbonyl
group of the reducing sugar and results in covalent bonding.
• Glucose and, occasionally, other reducing sugars (e.g., galactose) are present in
culture media, and glycation can occur during the upstream cell culture process.
• The methods that can be used to assess glycation include Boronate affinity
chromatography, MS of intact proteins and peptide map coupled with tandem mass
spectrometry (MS/MS).

Disialylation:

• For glycoprotein therapeutics (e.g.,erythropoietin), sialic acid plays a critical role in

biological activity and/or in in-vivo half life.
• Loss of sialic acid can occur slowly during storage under stability studies. Glycan
mapping can be used to monitor sialic acid loss.
Stability testing protocol

• Release and stability testing are a critical part of the overall control strategy
employed to demonstrate the quality, purity, safety, and potency of biological
products.
• At the time of submission of a marketing application for a new biological entity, the
routes of degradation for the molecule should be well understood.
Stability Indicating Profile:

• ICH Q5C recommends that “on the whole, there is no single stability-indicating
assay or parameter that profiles the stability characteristics of a
biotechnological/biological product.
• Consequently, the manufacturer should propose a stability-indicating profile that
provides assurance that changes in the identity, purity and potency of the product
will be detected”.
• Due to the number of attributes that might be tested, care should be taken to select
only those that are impactful.
Assay Selection:

a) Physiochemical Integrity

• Methods should be selected that are capable of detecting changes that may result
from chemical degradation, aggregation, and fragmentation.
• Analytical methods routinely employed to monitor purity include high resolution
chromatography methods (SEC, reversed-phase chromatography, ion exchange
chromatography), gel electrophoretic techniques (reducing and nonreducing gel
electrophoresis, isoelectric focusing), and peptide mapping.
b) Biological Activity

• The potency method (or methods) selected and included in the marketing
application should be relevant to the mechanism of action (MOA) of the product or
at least a surrogate assay that has been bridged to an assay that reflects MOA.
• The three main types of methods for measuring biological activity include binding
methods, cell-based bioassays, and animal-based assays.
• Binding methods (e.g., enzyme-linked immunosorbent assay (ELISA), surface
plasmon resonance) provide a measure of an antibody–antigen interaction.
• Cell-based bioassays measure a defined biological effect in vitro (the exact
mechanism being specific to the molecule in question)
• Animal-based methods measure the biological effect in vivo but are rarely used for
routine lot release or stability assays due to the challenges in performing such testing.
• Potency test methods should include either an international reference standard or a
qualified in-house reference standard if an international standard does not exist.
• While bioassays should be included in stability protocols for therapeutic proteins and
vaccines, these may not represent very sensitive stability-indicating methods: (i)
biologicals such as monoclonal antibodies are relatively stable under recommended
storage conditions, and during their proposed shelf lives, their potencies may not
change significantly; and (ii) certain degradation pathways expected to occur during
storage in stability studies that can be detected by more sensitive assays may not
impact potency.
c) Quantity (Assay):

• For proteins, quantity is typically determined by UV absorbance at 280 nm using a

theoretical or experimentally derived extinction coefficient.
• However, there are factors that may cause the apparent protein content to change:
(i) leachable compounds from containers/closures that cause an increase
in UV absorbance at 280 nm;
(ii) evaporation occurring during the stability study if container closure
integrity is compromised;
(iii) generation of degradants that have higher UV absorbance at 280 nm
than the protein itself (e.g., byproducts of Maillard reactions) [35];
(iv) loss of protein in solution due to adherence to the inner surface of the
primary container (this can only be observed with very low
concentration products); and
(v) microbial contamination
d) Safety:

• Sterility testing should be performed as part of drug product stability testing to

demonstrate the safety of the product throughout its expiry period.
• Per current FDA guidance [36], sterility testing should be performed during drug
product release (as container/closure integrity testing cannot demonstrate initial
sterility), but at subsequent stability time points, container/closure integrity testing can
be performed in lieu of sterility testing.
• European authorities have provided similar scientific guidance: “sterility testing should
be performed at least at the end of shelf life but it can be replaced by testing of the
container closure integrity”.
• Commonly used container/closure integrity methods include the widely adopted yet
less sensitive methods such as dye ingress, vacuum decay, and microbial challenge, as
well as newer technologies such as high voltage leak detection and laser-based gas
headspace analysis
• When drug substance is sampled from full-scale containers at each time point,
inclusion of safety testing in the drug substance stability protocol is a valid approach
to assuring the material remains within acceptable limits over its shelf life Commonly
used safety tests for drug substance include bioburden, sterility, and endotoxin.

e) Other Product Characteristics:

• Visual appearance, pH, and subvisible particulates should be monitored during

stability testing.
• For lyophilized drug products, reconstitution time and moisture content should also be
included.
• A visual appearance test should constitute a measure of color and clarity and an
assessment of visible particulate
• For biologics/biotechnological products in particular, the control of
visible/subvisible particle levels is especially important because of the propensity of
proteins to form particulates.
• The primary method for quantifying visible particles remains visual inspection.
• Changes in pH may lead to chemical degradation of the product. When this occurs, it
may be indicative of excipient breakdown or of leachables from interaction of the
product with the container/ closure.
• Levels of subvisible particles for injectable products and parenteral infusions should
be routinely monitored as part of drug product stability testing.
• The most common technique for quantitating the amount of subvisible particles
greater than 10 µm in size is the light obscuration method.
• Subvisible particles of less than 10 µm in size have come under increased scrutiny
by regulatory agencies in recent years due to concerns around possible
immunogenicity
• For drug products contained in semipermeable container/closures, moisture loss
testing should be performed.
• This can be carried out by monitoring container weight after storage under low
humidity conditions.
Stability Conditions and Timepoints:

Long term Storage Conditions:

•The long-term storage condition represents the defined storage temperature at which the
product will be stored.
•When shelf lives of 1 year or less are proposed, the real‐time stability time points should
include monthly testing for the first 3 months and at 3‐month intervals thereafter.
•For products with proposed shelf lives of greater than 1 year, the real‐time stability time
points should include testing every 3 months during the first year of storage, every 6
months during the second year and annually thereafter.
Accelerated and stress storage conditions on formal stability:

• Q5C does, however, make a strong recommendation that accelerated and stress
studies be performed on both drug substance and drug product.
• Conditions and time points should be selected on a case-by-case basis.
• Thermal stress conditions also provide value for stability throughout clinical
development and into the commercial stability phase.

Table 12.2 Commonly used accelerated and stress conditions for biologics.

Longterm storage condition Accelerated storage condition Stress storage condition

−90 to −70°C, −50 to −30°C 2–8°C 23–27°C
2–8°C 23–27°C 38–42°C
Other stress Testing:

• While the actual conditions selected are usually established on a case-by-case basis,
stress conditions for a biological product typically include thermal stress, extreme
pH exposure, oxidation (chemical), shaking/shearing, multiple freeze–thaw steps,
and photolysis.
• Milder, more relevant light conditions that mimic manufacturing and healthcare
facility settings are more meaningful for understanding the light sensitivity of a
product during manufacturing, storage, and clinical/commercial use.

Batch Selection:

• At the clinical stage of product development, at least one batch representative of the
manufacturing process of the material to be used in clinical trials should be
evaluated
• During late-stage clinical development where manufacturing process and product
presentation is consistent with or similar to that proposed for commercial stage, it is
beneficial to increase the number of lots on stability to at least three in support of
shelf life in the marketing application if no major changes are to be made during later
development.

• Within a marketing application, stability data should be provided for at least three
representative batches of drug substance and drug product, and a minimum of 6
months of stability data should be included.

• Stability data derived from the pilot plant scale may, however, be provided in
regulatory files with a commitment to place the first three manufacturing-scale
batches into the long-term stability program.
• This allows for the possible use of clinical stability data to be leveraged to set
initial commercial-scale shelf life.

• Stability data should be provided in support of all aspects of intended use of the
material (e.g., if a drug substance may be held frozen or at 2–8°C, then stability
data must be gathered in support of drug product produced using drug substance
held at these conditions).

• Similarly, if multiple freeze–thaws of drug substance are to be allowed, stability

data must be generated to support the manufacture of drug product produced from
drug substance subjected to freeze–thaws.

• Stability data should also be provided in support of any proposed hold times for
intermediates.
Sample Container /Closure:

• At all stages of product development, the drug substance placed on stability should
be stored in containers representative of the actual storage containers used during
manufacture.
• The use of reduced-size containers is permitted, provided that the contact
materials and container/closure systems are identical to those used in manufacture.
• Drug product with different container orientations should be included in the
stability study to evaluate potential impact of protein/container interactions.
• Typically, vials are stored in both inverted and upright positions, while syringes
are stored horizontally
Stability/Shelf life specification:

• A primary goal of stability evaluations is to assign and support the shelf life of
investigational product and product to be marketed commercially.
• Stability evaluation is performed and shelf life is established for drug substance in
order to demonstrate that the bulk material can be stored at a predetermined storage
condition for a specified duration without impact to the quality of the final drug product
manufactured using this material.
• The European Medicines Agency (EMA) has described acceptable approaches for
assigning a shelf life to a biological/biotechnological product during clinical trials,
stating that “extension of the shelf-life beyond the period covered by real-time stability
data may be acceptable, if supported and justified by relevant data”.
• Accelerated and stress testing may be leveraged for this approach, with the objective
being to compare the degradation pattern and rates of the material manufactured
using the new versus the original process.

• The goal is to demonstrate that the stability determined for the existing process can
be leveraged to assign shelf life for the material produced in the new process, rather
than directly utilizing the accelerated data to extrapolate shelf life at the long-term
condition for the new process.

• Any shelf life assigned using this approach should, of course, ultimately be verified
with real-time stability studies performed on the product manufactured using the new
process.
• By the time of submission of a marketing application, the shelf life of
biological/biotechnological products should be supported by real-time stability data,
with a minimum of 6-month data available at time of submission as stated in ICH
guideline Q5C.

• Stability data generated for pilot-scale material can be used to support the shelf life
proposed in a marketing application, as long as the pilot-scale material can be shown
to be fully representative of the commercial-scale product, albeit at a reduced scale.

• Comparability for both degradation pattern and rates should be demonstrated

between the pilot-scale material and the commercial-scale material in this case. A
minimum of three lots of materials from each process scale should be evaluated in
such comparability studies.
 Approaches used to determine shelf life data for
Marketing Applications:

• Patient safety and drug efficacy should always be the top factors to consider when
setting stability specifications
• A combination of process capability, manufacturing data, clinical exposure experience,
criticality of the quality attributes (impact on safety and efficacy), and analytical
method capability are taken into consideration when setting stability specifications.
• Regulatory agencies typically prefer that sponsors take a conservative approach
intended to protect the patient rather than reducing business risk. A risk/benefit analysis
should be performed to justify the proposed shelf life and associated specifications.
Case Study for Establishing stability specifications

• This is the long term stability data

of three hypothetical drugs.

• The minimum specification for the

purity of the drug product is 96.0%
• The shelf life determined by the individual stability measurements is 24 months. Lots 1 and 3 fail the
purity specification at the 36‐month time point; therefore the shelf life is determined based on the last
time point for which each of the lots passed, which is 24 months.
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