It goes without saying that structural characterization is the cornerstone of understanding your molecule and so this characterization must therefore assess all facets of your product. The ICH Q6B guidelines serve to define the areas of investigation that must be performed to generate as full coverage as possible – at the primary sequence level via peptide mapping, through post translational modifications and glycan characterization, to higher order structure (secondary and/or tertiary structure), physicochemical properties and aggregation.
These well-defined analyses are the best way to show your molecule is as it should be from a structural point of view under “ideal” or “normal” conditions. That is all well and good of course, but further consideration needs to be given to how the molecule behaves under “non-ideal” or extreme conditions. In practice, to address this requirement, specific mechanisms are used to purposefully degrade the sample in order to investigate what degradants are produced and the chemical breakdown pathways that these forced degradation processes cause the molecule to proceed along.
With these degradation mechanisms, we now have a model to assess how the sample will behave under extreme conditions such as may be met during failures of shipment and packaging or other stages from initial manufacture through to final handling at the patient location.
Furthermore, with the knowledge of the structure of any product degradants that are generated through the different forced degradation processes, manufacturing procedures can be adjusted to improve product quality.
Forced degradation studies can also serve to identify potential points of fragility of the product to external conditions, which can be further examined in controlled stability investigations.
Samples that have been forcibly degraded are useful to employ during qualification and validation of method development procedures (this is noted in ICH Q2(R1) guidelines on validation of analytical procedures), where they can serve to demonstrate specificity of the method under evaluation.
Finally, forced degradation and the idea of assessing the similarity of degradation pathways as part of a biosimilar comparability exercise is mentioned in the FDA and EMA biosimilar comparability guidelines (1,2).
Since various different chemical pathways will take place through the different degradation processes, these can produce changes at the primary amino acid level, have an impact on post-translational modifications such as glycosylation (if present), higher order structure (disturbance of secondary and/or tertiary structure) and also induce aggregation of the active ingredient. This means that several different aspects of structure need to be investigated in any forced degradation study. It is therefore important to have analytical procedures to hand that can provide structural information across this breadth of structural features.
Forced degradation studies should look to identify the nature of the modification(s) resulting from product stressing but should also attempt to localize its/their positions within the protein chain to the extent possible. Todays breed of mass spectrometers are eminently capable of performing this task since they have high sensitivity, allowing detection of low levels of degradation products. Instruments such as the Q-TOF geometry type of mass spectrometer are able to generate real time higher energy fragmentation data of peptides, including those peptides chemically modified by the stress conditions, in parallel with generation of low energy intact mass data.
Since fragmentation of peptides occurs across the peptide bonds in the amino acid backbone peptide mapping data can allow identification of which amino acid(s) are modified in any particular peptide that is exhibiting a change in expected mass as a result of the degradation conditions employed. For example, a peptide may be shown to be 16Da heavier in mass than predicted based on its amino acid sequence during a photostability study. Assessment of the high energy data derived fragmentation information from this peptide could show fragment ions consistent with an oxidized Tryptophan residue in the sequence, thus confirming the site of the oxidation.
There are no definitive guidelines for the precise methodology that should be employed for forced degradation studies of biopharmaceuticals, but in broad terms, high pH, low pH, oxidative, thermal and light stresses should be considered along with other more mechanical forms of stressing such as freeze/thaw and shaking. The precise nature of the degradative techniques used will depend not only on the nature of the sample in its final solution but also on whether the sample is a solution or a powder in its standard product state. So, conditions should be carefully selected on a case-by-case basis.
In essence, forced degradation of samples provides essential structural information both in terms of insight into the nature of the sample itself but also from a manufacturing perspective. This is important for any product but serves to provide extra valuable product information in a biosimilar comparability exercise. Results from forced degradation studies are used to establish a stability-indicating profile and will form an integral part of any regulatory submission.
For the analytical chemist, forced degradation studies can also give insight into which analytical methods are most reliable and sensitive to the specific changes induced, which allows development of an appropriate analytical package. Hence, analytical CROs are ideally placed to perform investigations into the various aspects of structure that need to be considered in a forced degradation study.
Please contact our scientists today who will suggest the best study plan for a forced degradation study of your molecule.
