The peptide drug market has rapidly expanded and is expected to generate in excess of $50 billion for manufacturers. While synthetic peptide synthesis is a cost-effective approach for producing these drugs, regulatory agencies are concerned that impurities resulting from the manufacturing process could introduce an unwanted immune response. Impurities can result from changes in the sequences due to deletions, insertions, substitutions, modifications and other impurities related to the synthetic production.
The FDA recently released a new draft guidance defining the equivalence of a rDNA peptide product and a synthetic peptide product enabling generic manufacturers of peptide drugs to file an Abbreviated New Drug Application (ANDA) for synthetic peptide drug products that refer to listed drugs of rDNA origin. Since the processes for manufacturing the generic and reference drug (RLD) are not equivalent, peptide drugs can be associated with impurities. The FDA draft guidance requires manufacturers to prove that synthetic peptide products do not contain impurities that have an increased affinity for major histocompatibility complexes and potential for engaging immune response, which may drive anti-drug antibody development.
Here we describe our PANDA assay that utilizes a combination of in silico assessment tools and in vitro assays to predict and validate the effect of peptide impurities on the immunogenicity of synthetic peptide drug products. In addition, we have developed a novel in-silico tool, the “What-if machine”, that mimics the process of synthetic peptide manufacturing and predicts the impact of impurities at any position in the sequence of a peptide drug.
Step 1. Immunoinformatics assessment: The potential of the DS to stimulate a T cell response can be rapidly assessed computationally using T cell epitope mapping algorithms. We use EpiMatrix for this purpose and focus on HLA DR (Class II) HLA binding predictions. In a typical DS analysis, the EpiMatrix algorithm is used to screen the primary amino acid sequence of the DS and its impurities, for the presence of HLA DR ligands, which can be considered putative T cell epitopes. Discriminating between potential inflammatory “T effector” epitopes and regulatory “T reg” epitopes is performed with a second algorithm, known as JanusMatrix. The latter algorithm identifies putative Treg epitopes, defined as HLA/epitope complexes that present a human-like outer contour (TCR face). HLA/epitope complexes that do not present an outer contour (TCR face) that is ‘human-like’ are more likely to drive effector T cell response. Following assessment of T cell epitope phenotype, the next step is to combine the scores for effector and regulatory T cell epitope content, providing an overall assessment of immunogenic potential of the DS and impurities. The resulting Treg adjusted EpiMatrix scores are highly correlated with immune responses in vivo.
Step 2: In vitro - HLA binding: The DS, its impurities, and peptides representing predicted T cell epitopes can be evaluated for binding to human HLA in assays that measure binding affinity in dose-ranging studies, in vitro. HLA binding is used to confirm in the in silico analysis and inform the design of in vitro assays (see step 3).
Step 3: In vitro assay – Teff Assay: Measurement of de novo T cell response: Cell culture protocols have been developed to emulate in vivo conditions that support differentiation of naïve T cells to effector T cells by antigen stimulation with biologics or their constituent T cell epitopes. In vitro stimulations using the biologic drug and human peripheral blood cells (PBMC) allow for natural antigen processing of the DS and other product components including impurities. In vitro assays using predicted epitopes derived from the DS product impurities provide information about the ability of these defined sequences to drive a T cell response using human T cells. At the end of the culture period, T cell phenotype and/or function are characterized in assays that measure the magnitude and quality of effector T cells that have potential to drive ADA development. Treg Assay: Co-incubation of the putative Treg epitopes with known T effector epitopes (such as Tetanus Toxoid-derived T effector epitopes) allows the assessment of bystander suppression. Bystander suppression is a feature of Treg epitopes.
In summary, in silico and in vitro assessment of novel T effector (inflammatory) and Treg (suppressive) epitopes is necessary to best evaluate the impact of novel impurities on the immunogenicity risk in humans. In addition to the standardized tools described above, we will describe novel in silico methods that have been developed to anticipate well-known synthetic peptide impurities (The What If Machine). A case study of Taspoglutide will be provided to illustrate these well-established (and more recently developed) Immunogenicity Risk Assessment methods.