Creative Biolabs can thoroughly assess the chemical modifications and post-transcriptional modifications (PTMs) of the drug candidates by our CreDA™ platform to identify the undesired PTMs that may have negative effect on the potency and in vivo immunogenicity.
Modifications in the chemical composition of biologics, whether by cellular processes or chemical reactions, can result in a complicated level of product heterogeneity, which may have negative effect on the potency and in vivo immunogenicity. Therefore, developability assessment in the early stage of drug development should figure out what modifications are likely to happen to the candidate that could compromise its performance. And critical quality attributes (CQAs) that are associated with chemical stability and undesirable PTMs should be controlled and monitored before a candidate is further moved to the process development. Below, we illustrate common chemical modifications and PTMs that will be assessed by our developability assessment service. Monoclonal antibody is one of the most common biopharmaceutical product class and can experience a wide range of modifications, thus it will become the focus of our attention.
Fig.1 Chemical stability and PTMs assessment.
Deamidation is a chemical process in which an amide group in the side chain of asparagine or glutamine is removed or converted to another functional group. Typically, asparagine is converted to aspartic acid or isoaspartic acid. Antibody deamidation could cause charge heterogeneity and affect the potency if it is located on the binding surface such as CDRs. Besides, deamidation may result in protein fragmentation, aggregation, or even increase the risk of immunogenicity. If structure data of the candidate is available, which allows a complete assessment of conformational flexibility and exposure of amino acid residues, the most accurate prediction of potential deamidation sites is guaranteed by us.
Antibody residues, such as histidine, methionine, cysteine, tyrosine, and tryptophan, are susceptible to oxidation caused by reactive oxygen species (ROS). Oxidation can generally be classified into two categories: site-specific metal-catalyzed oxidation and non-site specific oxidation. Methionine and tryptophan, to a weaker extent, are more susceptible to oxidation in non-site specific reaction. Methionine is dominantly sensitive to free ROS and tryptophan to light induced oxidation. The extent of sensitivity lies on solvent accessibility of the side chain, and residues buried in the protein core are relatively less sensitive or need longer reaction time. Antibody oxidation can occur during production, purification, formulation or storage stages, and the biological activity and stability may be negatively influenced by the introduction of oxidation. Combined with primary amino acid sequence and structural analysis, the solvent-exposed methionine and tryptophan will be carefully checked during our developability assessment to select candidates with minimized oxidation potential. If no alternative candidates are available, antibody re-engineering will be performed to remove the undesirable oxidation sites.
Glycosylation is a cellular process which can result in product heterogeneity and instability. Proper glycosylation, on one hand, is important to confer specific biological characteristics to a given biopharmaceutical, including its potency and pharmacological properties, on the other hand, can be a determinant factor in the folding and assembly of a product, and often defines other key attributes, such as stability, solubility, and immunogenicity. However, the incorporation of unintended glycan structures in or near CDRs may occlude the binding region of the product or introduce steric hindrances that could negatively impact binding affinity. Glycan structure can vary in branching and composition, thereby introducing further heterogeneity that could potentially impact product identity and other CQAs. Furthermore, the presence of non-human glycans is a known risk for hypersensitivity and anaphylactic reactions to biopharmaceutical products. Accordingly, evaluation of the glycosylation sites and glycosylation level is an important component of the developability assessment in order to select out candidates with desirable glycosylation.
Aspartate isomerization is the non-enzymatic interconversion between aspartate and isoaspartate. Aspartate residues with a succeeding glycine or proline (the “DG” and “DP” motifs) are more liable to isomerization. Isomerization could lead to charge heterogeneity of antibody, and if it occurs in the binding area such as CDRs, the antibody function may be significantly affected. Besides, in acidic environments, the peptide bond C-terminal to aspartate is susceptible to fragmentation, which will potentially increase the risk of in vivo immunogenicity. For candidate selection, we recommend avoiding those with potential isomerization motifs in the CDRs where possible. If no alternative candidates are available, antibody re-engineering will be performed to remove the undesirable isomerization sites.
Free cysteine residues
The presence of free, solvent-exposed thiol groups from cysteine constitutes one of the chemical instabilities with the highest risk for biopharmaceutical products. The reactivity of these groups can potentially promote disulfide scrambling, protein misfolding and aggregation, as well as increase the risk of immunogenic reactions. Besides, unwanted reactions of thiol group with other molecules in the environment may be introduced. In antibodies, free cysteines are most likely located in CDRs and can cause low productivity. They can be easily chemically modified before antibody reaches its antigen in vivo and may result in an altered binding capability. As part of our CreDA™ service, the candidate sequences will be scanned in silico and searched against an internal database to locate free cysteines and conserved disulfide bonds. Candidates with high-risk free cysteine residues will be de-selected or engineered to remove the thiol groups.
Chemical glycation can occur in a product due to reaction with sugars (present in media) during bioprocessing, and can potentially affect identity, stability, immunogenicity, or other CQAs of the biologics. Potential susceptible sites for glycation (i.e. solvent-exposed Lys) can be predicted by the in silico module of our CreDA™ platform. Moreover, forced glycation studies could also be performed to not only confirm the predicted susceptible sites but also help define the magnitude of the problem, as well as determine the environmental conditions that promote or prevent its occurrence.
C-terminal lysine cleavage
C-terminal lysine cleavage is a common PTM and occurs in the cellular process mediated by basic carboxypeptidases. The heterogeneity of the antibody charge and mass is mostly caused by the C-terminal lysine processing, resulting in a mixture of isoforms bearing one, two or zero lysine residues. Although it has been reported that C-terminal lysine variant antibodies exhibit the same potency and therefore, the biological relevance is minimal, maintaining consistent C-terminal lysine levels in therapeutic antibodies has been a process developmental target for product consistency, and each species should be monitored and kept within predefined limits.
Pyroglutamate formation is a kind of PTM occurring in proteins with an N-terminal glutamine or glutamate residue. The light or heavy chains of antibodies owns an N-terminal glutamine or glutamate, so the formation of pyroglutamate is very common. Glutamate conversion to pyroglutamate is unlikely to cause a safety issue. However, the N-terminus in an antibody lies close to the CDRs, and the charge variation may influence the binding affinity. Accordingly, the potential for pyroglutamate formation should be taken into account during the candidate selection stage, to get hold of the potential risk and provide information for process optimization.
N-terminal cyclization of a protein can happen through the nucleophilic attack of the N-terminal amine on the second carbonyl group of the backbone, ending up with diketopiperazine (DKP). N-terminal cyclization prevents Edman sequencing and can cause mass and charge variants which should be controlled and monitored. The N-terminal cyclization can be predicted by detection of N-terminal glycine-proline motifs.
Equipped with the advanced analytical instrument, Creative Biolabs has the ability to detect all kinds of post-transcriptional modifications occurred in the biologics (mainly antibodies), aiming to find out the potential undesirable modifications that may influence the drug potency or in vivo immunogenicity. If you are interested in our service, please contact us for more information and a detailed quote.