The Quality by Design paradigm demands an enhanced understanding of processes and products. Biotech therapeutic products, being complex molecules, require a robust analytical platform to serve as the foundation for commercialization activities such as the definition of critical quality attributes. This platform is generally a combination of orthogonal, high-resolution techniques that together provide definition to the product and the process. This article presents recent developments in four analytical applications, namely disulfide linkage analysis, glycan analysis, analytical ultracentrifugation, and flow injection protein analysis.
Determining CQAs and developing methods suitable for their measurement is an intricate and evolving science. This stems from the inherent complexity of biological macromolecules. A perfectly pure biological product is in itself challenging to characterize, typically comprising a chain of hundreds of amino acids and associated glycan subunits. This chain is sometimes linked within itself or to other chains through disulfide bonds, and then folded into a discrete secondary and tertiary structure.3–6 Quaternery structures also exist, from dimerization up to potentially large numbers of noncovalently or covalently linked subunits. Furthermore, manufacturing processes never achieve 100% purity, resulting in the need to measure and control product-related variants and product- and process-related impurities.
Product-related variants closely resemble the desired product and have a potency and safety profile equivalent to those of the product itself.7 Typical examples include minor post-translational modifications such as C-terminal processing, N-terminal variants, and deamidation. Product-related impurities, however, do not resemble the desired product with respect to safety and efficacy. Examples include aggregates and highly truncated forms. Process-related impurities include host cell DNA, host cell proteins, and raw materials from the process. A complex array of analytical methods is necessary to adequately characterize both the product and process.8 These methods must be developed and then qualified and validated for their intended use.9
A NEW METHODOLOGY FOR DETERMINING DISULFIDE BONDING PATTERNS
Regulatory agencies expect disulfide connectivity to be determined as part of product characterization.7 Contemporary methods for elucidating bonding patterns between closely-spaced cysteine residues include cyanylation-induced cleavage after limited reduction and limited reduction and alkylation followed by mass spectrometric analysis.10,11 The latter approach was successfully applied to a recombinant IgG2, revealing subspecies of disulfide variants.12 The main limitation of the reduction step inherent in both these approaches is the difficulty in finding conditions that allow for controlled reduction of specific disulfide bonds.
Recently, the analysis of mass spectral fragmentation patterns of disulfide-linked peptides has been used to elucidate disulfide linkages.13 However, the applicability of this approach has been limited to fairly simple systems, and not to the highly complex linkage patterns of the recently reported IgG2 variants.14
To overcome the above limitations, we have recently developed an innovative methodology that permits rapid determination of the connectivity between closely-spaced or adjacent cysteine residues in disulfide-linked molecules, and can be readily applied to complex monoclonal antibodies (MAbs) such as those in the IgG2 class. This methodology uses a unique combination of traditional Edman chemistry with mass spectrometric detection. Optimization of the sample processing for Edman chemistry played a crucial role and provided predictable structures for facile interpretation of mass data.
The characteristic residual and leaving species may be readily differentiated by mass spectrometry, and their identities may be confirmed by standard fragmentation analysis. If a mixture of disulfide structures for a given variant is present, it may not be possible to differentiate them from the observed leaving groups. However, the residual groups are diagnostic, and may even provide a means for a semiquantitative measure of their relative abundance, based on their relative signal in either the mass spectrometry (MS) or ultraviolet (UV) traces. Additional rounds of coupling, cleavage, and liquid chromatography–MS (LC–MS) analysis provide a further means for confirming structures to a greater level of detail, and also demonstrate the absence of scrambling during the chemical processing steps.
We have successfully applied this approach to the well-characterized insulin molecule and to a MAb where we identified new substructures of IgG2 variants, including the presence of intra-chain disulfide linkages in the hinge region.5 Details of this approach will be the focus of upcoming publications.