The US FDA’s Office of Biotechnology Products (OBP), part of the Center for Drug Evaluation and Research (CDER), has recently highlighted the rising usage of imaged capillary isoelectric focusing (iCEF) and imaging detection capillary isoelectric focusing (icIEF) for charge variant analysis during process development of biologic drugs, as well as mass spectrometry (MS ) for glycan analysis. IBO had the opportunity to discuss the usage of these technologies with Marjorie Shapiro, PhD, supervisory biologist, Division of Biotechnology Products Research and Review I, OBP, Office of Pharmaceutical Quality at the FDA’s CDER.

 

Capillary Isoelectric Focusing for Charge Variant Analysis

The Chemistry, Manufacturing and Controls sections of investigational new drugs (INDs) and biologics license applications (BLAs) for biological products are reviewed by the OBP and regulated by the CDER. As part of regulatory oversight, data generated by technologies such as iCEF and icIEF are used in drug applications. Such technologies’ use spans both research and QA/QC applications. “We strongly encourage use of state-of the-art methods for characterization of products and implementation as QC methods when feasible,” Dr. Shapiro said. “Our experience is that state-of-the-art methods are adopted as characterization methods before they are used as QC methods, which must meet stricter cGMP standards.”

iCEF and icIEF  have gained widespread acceptance as analytical techniques for supporting BLAs, largely due to the fact that capillary-based techniques are generally more reliable than gel-based techniques and can be amended to meet QC requirements, according to Dr. Shapiro. “Post-translational modifications (PTMs) can influence the isoelectric point of a protein, resulting in a range of charge variants,” she explained. “Many PTMs do not affect the biological activity of a protein, but some may reduce potency or make the protein more immunogenic; therefore, it is important to understand the PTMs that contribute to each charge variant to establish an appropriate control strategy for charge variants overall, and for specific charge variants that could affect the safety and efficacy of a product.”

“A capillary-based method consistently identifies the same number of peaks on the electropherograms run to run.”

As Dr. Shapiro stated, capillary-based methods are now often used instead of gel-based methods for the identification of both protein size and charge variants due to greater resolution and reproducibility. “Compared to the older gel-based methods of separation, capillary-based methods more readily provide quantitative results for the main peak and product-related variants, including variants that may occur at low levels,” she said. “In addition, a capillary-based method consistently identifies the same number of peaks on the electropherograms run to run. For example, an IEF gel might resolve 9 bands one time and 5 bands from the same lot the next time it was run, or the number of bands might vary lot to lot.” According to her, the number of bands that are detected in the reference standard could also vary between the gels. “If a new peak was detected by a capillary-based method and system-suitability criteria were met for that run, it would be clear that either there was some degradation of the lot or, if it is a new lot, the new lot is different from other lots and an investigation would be opened to understand this discrepancy.”

 

MS for Glycan Analysis

The FDA CDER has also emphasized the rising usage of MS for glycan analysis during process development of biologic drugs as the analysis of these drugs became more comprehensive. As Dr. Shapiro shared, previously when the Agency reviewed IND applications for therapeutic monoclonal antibodies (mAbs), monosaccharide analysis was the key technique used to assess glycans. “It wasn’t clear what significance, if any, there was between differences in the molar ratio of a specific monosaccharide when comparing lots manufactured before and after manufacturing changes,” she explained. “Our understanding of the importance of specific glycan structures at that time was limited to the knowledge that mAbs with glycans lacking galactose had reduced complement-dependent cytotoxicity (CDC) activity.”

“The ability of MS to identify major, as well as minor glycan species will help advance our knowledge of structure function relationships of therapeutic proteins.”

Although there are other methods that can analyze glycan structures, MS has the capability to identify and quantitate many specific, as well as minor, glycan species, Dr. Shapiro noted. “In parallel with advances in other biological activity methods for mAbs, such as antibody-dependent cellular cytotoxicity (ADCC), an understanding of specific glycan structures, such as those that lack fucose, have high mannose or terminal sialic acids, led to an understanding of the role that specific glycan structures have on ADCC activity and potentially other Fc-mediated effector functions,” she said. “This knowledge enabled sponsors to generate specific structure-function relationship models that suggest small differences in levels of afucosylated glycans can have a large impact on ADCC activity.  Experimental data using mAbs with different levels of afucosylated glycans confirmed these models—it would not surprise me if additional knowledge demonstrating a role of other glycan structures for antibody effector functions is discovered.”

Glycan structures additionally play a significant role when it comes to biological activity of therapeutic proteins, such as erythropoietin and replacement enzymes, as Dr. Shapiro explained. “The glycans on these proteins are more complex than those on antibodies,” she noted. “The application of MS to characterize these complex glycan structures, as well as other methods for the control strategy of these products, are also routinely performed. The ability of MS to identify major as well as minor glycan species will help advance our knowledge of structure-function relationships of therapeutic proteins.”

While MS for glycan analysis is not used as a QC method in current practices, it is a highly valued technique for testing of biologic drugs, such as biosimilars. “[MS] plays a major role in the characterization of products, for demonstrating comparability of a product before and after manufacturing changes, and in establishing analytical similarity between a proposed biosimilar product and the reference product,” Dr. Shapiro said. “Other methods for glycan analysis, such as 2-AB HILIC-HPLC or UPLC, are used as QC methods as part of the control strategy.”

 

New Analytical Bioprocess Techniques

The FDA CDER has promoted the use of new analytical techniques for bioprocess development through its Emerging Technology Program. The Program aims to foster the adoption of cutting edge techniques for pharmaceutical product design and manufacturing. As Dr. Shapiro stated, examples of innovative technologies for biologics testing include the MS-based multi-attribute method (MAM).

“With enhanced knowledge of a product, the most appropriate control strategy can be developed.”

It would be a detriment to limit new, innovative analytical techniques, such as (MAM)  to solely new product development programs, Dr. Shapiro explained, as the OBP encourages introducing innovative and improved methods for legacy products as well. “We believe that using new methods, especially for impurity methods, are valuable for detecting unexpected impurities that could impact the safety or efficacy of a product,” she noted. “If a new method is intended to be a release/stability method, it needs to undergo appropriate validation, and both methods should be used for release and stability testing until adequate data are collected to establish a bridge between lots tested by the previous method and lots that will be tested by the improved or new method. Therefore, we recommend that sponsors store retain samples of historical lots under conditions that will minimize degradation.”

Thus new testing methods can directly impact previously manufactured products. Dr. Shapiro explained that if a “new” impurity is picked up by the novel method but the impurity is also present in legacy lots, the OBP would determine that the impurity is not related to the new product, but is actually a newly detectable product-related impurity. “[In this case], the sponsor should characterize this product-related impurity to understand its potential impact on safety and efficacy of the product,” she continued. “OBP would work with the sponsor to develop an appropriate control strategy, if necessary.”

Generally speaking, a well-characterized therapeutic protein can help pinpoint quality attributes that may affect the efficacy, safety, pharmacokinetics or immunogenicity of a product, Dr. Shapiro noted, and newer methods are valuable for identifying the degradation pathways of products. “With this enhanced knowledge of a product, the most appropriate control strategy can be developed, and sponsors will be able to more readily control and ensure lot-to-lot consistency of their products,” she said. “The consistency of product quality attributes of a therapeutic protein ensures that patients can expect each dose they receive of the product will work the same every time.”