What are the essential steps you would have to take to comprehensively characterise a protein therapeutic?
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Ashleigh Wake
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The analytics chosen should be capable of fully elucidating the physiochemical, structural and biological features of the molecule, as well as quantifying purity and levels of impurities. More so, the testing should have a strategic approach – meaning that it should include consideration of features which are of greatest potential relevance to efficacy or safety of this specific molecule. For example, prior to finalising a testing programme designed to assess purity/impurities it should be fully understood what the potential impurities are and thus confirm that the methods applied are fully capable of identifying, and if appropriate, quantifying these species. This particular aspect should be achieved through the application of forced degradation studies using extreme environmental factors to aid in the prediction of likely, or possible, degradation pathways.
Which regulatory guidelines are pertinent to this area?
Regulatory agencies throughout the world have issued documents detailing the general expectations for characterisation of biopharmaceuticals such as the EMEA Biological Medicinal Products, last updated May 22, 2015. In terms of the considerations during characterisation the guidance in ‘ICH Q6B; Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products,” adopted May 2001, gives perhaps one of the most detailed descriptions of what should be included or considered during characterisation.
Explain the concept behind the ‘orthogonal technique’ approach?
Given the complexity of the biologic molecules under evaluation, the application of ‘orthogonal’ or alternative techniques are often required in order to provide confidence in results but also to provide analytical packages to support product development from R&D through to manufacture. For example, considering an evaluation as simple as intact molecular weight, mass spectrometry provides perhaps the most accurate measure of molecular weight and this approach is ideal when considering analysis of a lyophilised material typical of an early phase product. Once the material is formulated as in the final product the excipients may be incompatible with a mass spectrometry endpoint and thus have to be removed prior to analysis. This introduces the risk of affecting the structure or properties of the active biologic in some way. Using an alternative technique such as SEC-MALLS or SDS-PAGE could be used in preference.
What are some of the hot topics concerning characterisation currently?
There are multiple areas which could be considered as hot topics at the moment, such as assessment of aggregation, post translational modifications and protein interactions. However, given the huge advances in analytical capability and information obtainable all topics can be considered as ‘hot’ as molecules can in general be characterised to a greater extent than previously possible.
Protein aggregation remains to be a hot topic in the industry. How would you conduct an aggregation study?
There are multiple methods applicable to the assessment of aggregation; this is one area in which orthogonal testing is a must to ensure the validity of the result. Typically one stationary and one dynamic approach is applied such as Analytical Ultracentrifuge (AUC) or Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS) or SDS/NATIVE PAGE/western blot.
In the course of a protein therapeutic stability study what would be the key attributes that you would monitor as stability indicating?
Testing needs to be performed to assess the levels of purity and impurities using methods that have been shown to be stability indicating in a way similar to what would be expected for small molecules. This includes the assessment of fragments and aggregates etc.
In addition the biological potency of the molecule throughout the storage needs to be established along with standards test such as pH, appearance sterility, bioburden and endotoxin etc. Specialised testing looking at certain pertinent structural features, such as secondary or other higher order structural features may also be required. Where these extended analytics are applied testing is usually performed at selected time points only.
According to regulatory guidelines for biosimilars extensive comparability testing will be required to demonstrate that a biosimilar has a comparable profile in terms of their quality, safety and efficacy as the reference product. What are the aims of these studies?
To establish that the physiochemical, structural and biological aspects of the biosimilar are comparable to the innovator materials. This is absolute importance to establish that the biosimilar is infact a biosimilar and not a new biological entity (NBE). There is unavoidable heterogeneity observed between different batches of innovator materials themselves therefore when comparing a biosimilar to an innovator, multiple batches of each are required such that comparability can be based on determination if the variability of features within the biosimilar falls within the variability of the same aspect in the innovator.
How would you perform them? Which analytical techniques would you use?
A full suite of analytics as used in characterisation are required, including assessment of physiochemical features (such as mass, size, purity/impurities, isoform distribution, concentration), structural features (such as amino acid distribution and sequence, terminal sequence confirmation, disulphide bridge assessment, spectroscopy studies) as well as assessment and comparison of biological potency.
What is the importance of carrying out cell-based potency assays as part of a comparability study?
Bioassays are used to assess the potency of the molecule. This is a fundamental feature for establishing comparability as it is not enough for the chemistry of the molecules to be comparable but the species such also be shown to have the potential to produce the same response
Monoclonal Antibody therapeutics (mAbs) and Antibody drug conjugates (ADCs) appear to be very promising for the future. What are the specific complexities of a characterisation a mAb?
Generally all issues to do with mAbs are related to their size which is around the 150KDa region. This makes all analytics more complex.