Anette Skammelsen Schmidt



	Forced degradation studies are performed by means of various stressing agents such as pH, temperature, light, chemical agents (e.g., oxidizing, deamidating agents, etc.), and mechanical stress to speed up the chemical degradation, physical degradation, or instability of a molecule. Currently, there are no industry guidelines available defining how to perform forced degradation studies for biopharmaceuticals. The guidelines only provide useful definitions, general comments, and a rough concept about degradation studies (1–5). Strict guidelines with specific ranges or exact conditions for forced degradation studies are not necessarily possible, as every molecule is different, and certain freedoms for selecting stress conditions for biopharmaceuticals are inherent (4, 5). Hence, conditions should be carefully selected on a case-by-case basis (3).

	Regulatory guidance documents specify the following expectations on forced degradation:

		The manufacturer should propose a stability-indicating profile that provides assurance that changes in the identity, purity, and potency of the product can be detected (2, 3).

		Results from forced degradation studies will form an integral part of the information provided to regulatory authorities (4, 5).

	Furthermore, studies exposing the biopharmaceuticals to stress conditions may be useful in determining whether accidental exposures to conditions other than those proposed (e.g., during transportation) generate changes in the molecule. Stress studies are also useful for evaluating which specific test parameters may be the best indicators of product stability and should be monitored under proposed storage conditions (3).


	Forced degradation study is defined as an intentional breakdown of a molecule to an appropriate extent by means of various stressing agents (including mechanical stress) to speed up the chemical and physical degradation and instability of a biopharmaceutical. A forced degradation study can give a range of information regarding the likely degradation products of a specific biological drug. This information can be useful for many purposes, and can help to establish the degradation pathways and the intrinsic stability of the molecule. Challenging the analytical procedures helps validate the method’s stability-indicating power (4, 5).

Prior to performing a forced degradation study, the goal of the study needs to be defined. Several purposes might be addressed in one study. When relevant, a forced degradation study can be performed at different development stages. 

 shows examples of the various reasons that forced degradation studies are performed.

	Degradation products for biopharmaceuticals may be either product-related substances or product-related impurities, as some degradation products may retain biological activity (1–3). An example of this is illustrated in 

 and describes a situation in which oxidation is not associated with a decrease in activity.

Degradation pathways of biopharmaceuticals


	Biopharmaceuticals can usually degrade in many different pathways following different kinetics. The extent of stress needs to provide a measurable change and confirm the most relevant degradation pathways. Too much stress, however, might form secondary degradation products not seen in formal stability studies, and the level of stress might not reflect actual potential stressors. An extent of degradation of approximately 5–20% is assumed to be suitable for most purposes and for most analytical methods. An adequate level of stress should be carefully selected on a case-by-case basis (3).

	The selection of the degradation pathways to be investigated during forced degradation should be based on known and anticipated degradation pathways-as well as prior knowledge from similar molecules, if such knowledge exists. The degradation pathways are typically either physical (e.g., aggregation) or chemical (e.g., oxidation) in nature.

	Aggregation can be noncovalent in nature, such as an association of monomers that are dissociable at the right conditions (e.g., solvent, temperature). These noncovalent aggregates are mainly formed by denaturation and unfolding of the molecule, or by an interaction with interfaces such as liquid-air, liquid-solid, or even liquid-liquid. These associations are typically a result of mechanical stress such as shaking, stirring, rotation, pumping; freeze-thaw cycles; heating; or exposure to acidic pH.

	Aggregation can also be covalent in nature, such as chemical bonding between the molecules, and is non-dissociable during buffer change. These chemical bonds are often formed by rearranged disulfide bridges or other altered intramolecular bridges. They are typically a result of reactions of the amino acid residues with trace metals (copper or iron) or an incomplete reduction of the protein.

	Side chains of methionine, cysteine, histidine, tryptophan, or tyrosine residues are susceptible to oxidation, where methionine is the most reactive residue. Oxidation can alter the physicochemical properties of a protein, such as folding and subunit associations. The oxidation is mainly due to exposure to atmospheric O

 under conditions of light, heat, moisture, agitation, or to exposure to oxidizing agents. Deamidation is a hydrolytic conversion of asparagine or glutamine to a free carboxylic acid residue and is typically due to changes in pH, ionic strength, temperature, and humidity in the case of lyophilized proteins. The overall effect of a chemical modification of a single amino acid residue depends on its position in the protein and on the specific role the residue has in the functionality and active site of the protein.

	Photolysis by exposure to light involves a free radical mechanism that affects many functional groups (e.g., carbonyl groups). The free radicals can result in oxidation, aggregation, or peptide bond cleavage. The photolysis is due to exposure to photo-irradiation, which is typically in the form of ultraviolet irradiation.

	Hydrolysis (fragmentation) is a cleavage of peptide bonds between amino acid residues releasing smaller peptide chains. The peptide bonds of Asp-Pro and Asp-Gly are the most susceptible to hydrolysis. Hydrolysis is mainly a result from exposure to acidic or alkaline pH.

	Disulfide bridge exchange might cause incorrect paired disulfide bridges, which affects the tertiary structure of a protein. Such incorrect disulfide bridges might be a result of partial cleaving and reformation of disulfide bonds as a result from denaturing/reducing conditions (exposure to reagents such as GnHCl, urea, and 1,4-Dithiothreitol [DTT]) and oxidation of cysteine residues such as oxidation by Cu (II) or Fe (II) ions.

	Several biopharmaceuticals contain ligands or conjugates. Such bound moieties (e.g., acylation and conjugation) might be lost due to chemical or physical stress on the molecule.

Selection of materials for a forced degradation study


	When performing forced degradation studies, it is important to use a single batch of material. Forced degradation studies usually require a large amount of material. However, the material could be non-GMP, a test batch, or even an out-of-specification batch (if such is available), as long as the choice of batch is justified.

	All relevant sample types should be included in the forced degradation study. Drug product at both high- and low-dose levels can be included for drug product-specific methods. If the molecule is modified (e.g., by acylation, glycosylation, or conjugation), the inclusion of the intermediate is highly recommended to aid understanding of the changes seen in the underlying structure of the molecule. Solution/buffer blanks and controls (excipients) are included for evaluation of peak profile regarding occurrence of new peaks as a result of stress conditions. Always include reference samples in each experiment.

Selection of analytical methods for forced degradation studies

Due to the complexity of biopharmaceuticals, there is no single stability-indicating method that can profile all its stability characteristics (2, 3). The nature of biopharmaceuticals will dictate which test methods to use. In general, methods that are used in stability studies should be included in forced degradation studies, as well as methods that determine identity, purity, content, and methods for monitoring impurities. The methods should provide reliable data-as measured by a satisfactory selectivity between the main peak and impurities-an adequate intermediate precision, and be able to detect the change if/when it occurs. Examples of analytical methods to evaluate degradation pathways are shown in 

	Examples of common methods to employ for analysis of biopharmaceuticals during forced degradation are appearance (i.e., color, clarity, particulate matter); activity measurement; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE); microchip gel electrophoresis; size-exclusion high-performance liquid chromatography (SE–HPLC) (e.g., for protein content and aggregates); reversed-phase high-performance liquid chromatography (RP–HPLC) (e.g., for purity and specific impurities); isoelectric focusing (IEF)/imaged capillary isoelectric focusing (iCE)/ion-exchange HPLC (IE–HPLC) (e.g., for deamidated forms); peptide mapping; biological activity; and physicochemical analysis (e.g., differential scanning calorimetry [DSC], circular dichroism [CD], and fluorescence). Additional analysis can be employed based on the results obtained by the initially selected analytical methods.

Suitable conditions for forced degradation studies

All molecules can be degraded by some chemical or physical means. 

 shows examples of common stress conditions known to induce different degradation pathways for biopharmaceuticals. The conditions used in forced degradation have to be harsher than conditions used in accelerated studies. If the conditions result in no change, longer exposure time is recommended, rather than the use of a more extreme temperature. When selecting the relevant stress conditions, the following points must be considered:

		How many extra samples (for new methods and characterization) were used?

The total protein content should be measured for all samples (as shown in 

) to evaluate the presence of insoluble aggregates. As the determined total protein content is constant under the applied conditions, insoluble aggregates are not formed under these conditions. Conditions of high temperature and long periods of time, however, lead to high amount of high-molecular weight proteins (HMWP).Reference samples have to be placed next to forced degradation samples in order to evaluate the cause for an observed effect. All samples from a specific study need to be analyzed in the same analytical series to exclude the effect of possible analytical variation.

Forced degradation during the development phases


	Forced degradation studies can be performed in early development or late development depending on the purpose of the study and the amount of material available. The health authorities expect forced degradation studies to be carried out during development Phase III at the latest, but no guide or specific requirements exist about when to perform forced degradation studies.

	A forced degradation study will provide knowledge about the degradation pathways of the molecule. By performing such studies early in development, this knowledge about the molecule will be available for optimal process and formulation development. The degraded samples can aid the development of stability-indicating analytical methods by demonstrating if the current methods are sufficient to evaluate stability (e.g., use oxidized samples to develop method for determination of oxidized forms) and by identifying which test parameters are the best indicators of stability. Degraded samples are also useful during analytical validation, as they can be spiked in validation samples. However, a limited amount of material is usually available at the early stage of development and the analytical package might be incomplete. During development, the process steps and the formulation might change. Additionally, the analytical methods might change due to further optimization of the analytical conditions. Hence, forced degradation studies most likely need to be repeated or extended at a later stage of development. In conclusion, a limited forced degradation study should be performed as early as possible during development, and a more comprehensive forced degradation study during Phase III should be performed.

General evaluation of forced degradation studies


	Results from forced degradation studies should be presented graphically and should include compare plots for chromatogram methods. A result matrix is an excellent way to show results, as such a matrix will be able to indicate which forced degradation conditions resulted in changes for which degradation pathway. Statistical and kinetic tools should be used for evaluation of data when possible to aid the understanding of the degradation kinetics. A forced degradation study reveals the most important degradation pathways. Such pathways can, for example, be indicators of aggregation or the formation of specific impurities that could cause concern. The forced degradation study also indicates which analytical methods are most concerning and whether these methods are able to detect the change that occurs. In summary, degradation pathway studies can help investigators predict whether an analytical package is sufficient for a molecule or whether a need exists for the development of other analytical methods.

Specifications: New Chemical Drug Substances and Products

Specifications Test Procedures and Acceptance Criteria for Biotechnological/Biological Products

Stability Testing of Biotechnological/Biological Products

, Step 4 version (1995).
	4. ICH, Q1A(R2), 

Stability Testing of New Drug Substances and Products

Guideline On Stability Testing: Stability Testing of Existing Active Substances and Related Finished Products

 (London, October 2003).
	
	Figures courtesy of the author.


	Anette Skammelsen Schmidt, PhD, is senior research scientist, API analytical development, at Novo Nordisk A/S, Denmark.

 When referring to this article, please cite it as A.S. Schmidt, "Forced Degradation Studies for Biopharmaceuticals," 

Articles

The author addresses critical issues to consider prior to performing forced degradation studies and provides best practice recommendations for these types of studies.