Creative Biolabs has established a platform to predict and assess a series of post-translational modifications of antibody therapeutics. Deamidation of asparagine residues is one of the most common post-translational modifications occurring in therapeutic proteins produced using recombinant DNA technology, resulting in the conversion of an asparagine residue to a mixture of isoaspartate and aspartate.
The deamidation rate is influenced by three primary factors: pH, primary sequence and conformational effects. Antibody Exposure to alkaline pH results in an increased rate of succinimide formation due to greater deprotonation of the peptide bond nitrogen at higher pH values. Highest frequency for protein deamidation is the primary sequence with Asn-Gly, and intermediate frequency is Asn residues followed by a polar amino acid with a relative small side chain, like Ser, Thr and Asp. Asn residue followed by a hydrophobic amino acid with a bulky side chain has the lowest frequency for protein deamidation. The secondary and tertiary structure of protein can also alter the deamidation rate, as shown in two hotspots with the same primary sequence in the Fc regions of antibodies. The asparagine and its flanking residues must be solvent accessible and locate within a conformationally flexible region of the protein.
Asparagine deamidation could cause charge heterogeneity and hence affect the antibody function if it locates in a binding surface such as CDRs. Deamidated residues are likely susceptible to Aspartate isomerization or result in protein fragmentation, aggregation and immunogenicity. As deamidation is heavily caused by pH and process conditions, careful selection of process parameters to minimize the risks is indispensable. If crystal structure data is available, which allows the most complete assessment of local conformational flexibility and exposure of amino acid residues, combined with primary sequence information, Creative Biolabs guarantee to provide the most accurate prediction of deamidation.