Structural characterization is the cornerstone of understanding your molecule. The analyses described in the ICH Q6B guidelines are designed to assess many different facets of biopharmaceutical structure ranging from the primary sequence level, such as peptide mapping, to assessments of post translational modifications and glycan characterization, higher order structure (secondary and/or tertiary structure), physicochemical properties and aggregation. This is the best way to prove that your molecule has the expected structure under “ideal” or “normal” conditions. However, consideration also needs to be given to how the molecule behaves under extreme conditions and this is where Forced Degradation studies come in.
What is Forced Degradation?
During a Forced Degradation study, mechanisms are used to purposefully degrade the sample in order to investigate what degradants are produced and to understand the chemical breakdown pathways that the molecule takes when it is subjected to these stresses.
Why Should I Perform a Forced Degradation Study?
The data obtained from a Forced Degradation study may help you understand how your product will behave under extreme conditions such as those met during failures of shipment, packaging or other stages from initial manufacture right through to final handling at the patient location. Furthermore, knowing the structure of the product degradants (such as the formation of aggregates, see figure below) that are generated through different forced degradation processes means that manufacturing procedures can be adjusted to improve product quality, if required. 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.
Increasing Stress (e.g. temperature, pH)
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 and is therefore a key study to include during biosimilar development.
Instrumentation and Analytical Procedures for Forced Degradation Studies
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 and is therefore a key study to include during biosimilar development.
This means that several different aspects of structure need to be investigated in any Forced Degradation study. It is therefore important that the most appropriate analytical procedures are available to provide structural information across this breadth of structural features.
Forced Degradation studies should aim to identify the nature of the modification(s) resulting from product stressing but they should also attempt to localize its/their positions within the protein chain. Todays 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, data generated during the course of a peptide mapping analysis allows identification of which amino acid(s) are modified in any particular peptide exhibiting a change in expected mass (as a result of the degradation conditions employed).
Digestion of the protein using enzymes breaks the protein down into peptides. The enzymes used are chosen based on the theoretical sequence of the protein and a knowledge of how the enzymes will theoretically digest the protein. In this way, an informed choice of enzymes(s) can be made that should result in the best peptide mapping analysis.
For example, a peptide may be shown to be 1Da heavier in mass than predicted based on its amino acid sequence during a thermal stability study. Assessment of the high energy data derived fragmentation information from this peptide could show fragment ions consistent with an Aspartic acid residue in the sequence, where Asparagine has previously been shown to be present thus confirming the site of the deamidation.
On-line LC/ES-MS analysis of a tryptic digest of a protein. A: The upper two panels show a section of the total ion chromatogram and the extracted ion chromatogram for a deamidated peptide of interest. B: The lower two panels show the low energy mass spectrum demonstrating the presence of the deamidated peptide and the higher energy MSe data showing the fragmentation of that peptide. Signals observed are not only consistent with the peptide, confirming its detection but also demonstrate that deamidation has occurred exclusively on the first of the two adjacent asparagine residues.
Forced Degradation Parameters
There are no definitive guidelines for the precise methodology that should be employed for Forced Degradation although a recent article reports the findings of a survey on current Industry perspectives. 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.
In summary, Forced Degradation of samples not only provides useful structural information on the nature of the sample itself, but also helps with process development and manufacturing decision making. BioPharmaSpec is highly experienced in the design of Forced Degradation studies and the analytical investigations required to assess the various aspects of structure discussed above.
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