Ashleigh Wake began her 15 October 2020 “Ask the Expert†presentation by pointing out that peptide products are manufactured in a “regulatory vacuum.†Peptide-product developers must be strategic in designing characterization and quality control (QC) programs. Wake reviewed available methods and explored key considerations for developing phase-appropriate analytical controls.
Wake’s Presentation
Because peptides overlap small- and large-molecule drugs in size, regulatory expectations differ by product size and clinical indication. Thus, analytical programs should be designed around critical quality attributes (CQAs) that influence peptide-product safety and efficacy. Deep understanding of a peptide’s structure, physicochemical properties, impurity profile, and potential for interaction with excipients can help to delineate analytical requirements for different product-development stages.
Structural Analysis: Slight changes in structure can reduce a peptide’s efficacy and increase its immunogenicity. Amino acid analysis (AAA) can quantitate distribution of amino acids but cannot discern their sequence. For that, analysts need liquid chromatography with tandem mass spectrometry (LC-MS/MS) or Edman degradation. The former can confirm sequences, identify misincorporations, and map disulfide bond formation. The latter can confirm sequences from the N-terminus and distinguish isobaric amino acids.
Peptides can display high conformational flexibility in their secondary structure. To identify problems, analysts often pair circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which can ascertain percentages of alpha helices, beta sheets, beta turns, and unordered structures. Differences in CD and FTIR data should prompt further investigation using Raman or nuclear magnetic resonance (NMR) spectroscopy. To elucidate a peptide’s entire structure (e.g., to detect changes in sequence or assess comparability), analysts must apply NMR or X-ray crystallography.
Analyzing Aggregation: Peptides can aggregate nonspecifically in response to manufacturing and storage conditions, including interactions with bioprocess equipment and changes in temperature and pH. Aggregates range in size from dimers to polymers up to 1,000 kDa in mass.
Wake recommended selection of methods that can distinguish between peptides and small aggregates. Size-exclusion chromatography (SEC) is an ideal solution that many QC facilities can accommodate. SEC can separate small and large aggregates, sometimes after running multiple columns. Because SEC can induce aggregation, analysts should apply a complementary method such as analytical ultracentrifugation (AUC). Dynamic light scattering (DLS) is helpful in some contexts but can’t always distinguish monomers from dimers and trimers. Wake suggested using results from forced-degradation studies of aggregate forms (rather than data from peptide-spiking studies) to determine resolution between monomers and small aggregates.
Impurity Profiling: Product-related impurities account for the bulk of a peptide’s impurity profile. Forced degradation studies using LC-MS can help to establish impurity limits, but guidance for setting specifications differs by peptide size. ICH guidelines apply directly to peptides of <10 amino acids; larger products are evaluated by indication and immunogenicity potential. However, in silico modeling and in vitro approaches now can be combined to estimate a product’s immunogenicity. That strategy, Wake noted, has helped the generics industry to evaluate impurities that do not appear in a reference drug but do appear in a biosimilar product.
Formulation Concerns: Peptides can interact with formulation components: e.g., benzyl alcohol, a preservative, can initiate conformational changes, and polysorbate surfactants can induce antibody production. Similar concerns arise with container systems. Vial closures can compound aggregation. When such components are necessary, additional controls should be applied.
Questions and Answers
When should QC testing begin? Early in product development, QC testing needs to establish that a peptide is not prone to aggregation. Impurity profiles also help developers to determine appropriate process changes and control strategies.
When should peptide stability be assessed? Such testing must be finished by investigational new drug (IND) application filing, but robust evaluation before that stage helps to address aggregation, degradation, and product-related impurities. Analysts must ensure that tests are truly stability indicating within several storage conditions.
What questions do regulatory agencies ask most about peptides? Regulators want to know how well product-related impurities have been characterized. Even in the generics industry, regulators are insisting that products undergo intensive characterization testing before reaching patients.