Protein deamidation in biopharmaceutical manufacture: understanding, control and impact

Abstract

Understanding of product-related variants, such as variants with post-translational modifications, is an important part of biopharmaceutical development. Deamidation is a common post-translational modification occurring in biopharmaceutical proteins, affecting L-asparagine (Asn) and to a lesser extent, L-glutamine (Gln) residues. The rate of deamidation reactions are influenced by factors including protein structure (primary, secondary and higher structure), temperature and pH. In the vast majority of cases, deamidation is undesirable in biopharmaceuticals, and may lead to potential changes in protein structure, function, stability and immunogenicity. Measurement and characterisation of deamidated biopharmaceutical variants may be challenging, particularly with regard to quantitation of the two L-aspartate isoforms that are created, L-aspartic acid (Asp) and isoaspartate (isoAsp). Deamidation may occur intracellularly or during biopharmaceutical manufacture and storage, and must be understood, minimised and controlled, particularly in a regulatory context. Process control strategies that have been employed to date include alterations to fermentation steps, additives to cell cultures, chromatographic separation of charge variants and protein engineering to remove deamidation-prone Asn residues. However, the impact of deamidated forms of biopharmaceuticals should also be thoroughly studied, as they may not necessarily represent deleterious changes to the function of the molecule or the quality of the final product. This mini-review provides a summary of the current understanding of the origins, control and measurement of deamidation during biopharmaceutical development.

Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

“…30,45 However, peptide deamidation does occur in biological peptides and proteins, though it is most often associated with the polar non-charged residues of asparagine and glutamine, and not lysine as observed here. 46,47 Deamidation typically depends on multiple attributes such as sequence and 3D structure, and is thought to be a post-translational modification that increases in incidence with organism age. 47 …”

Deamidation Slows Curli Amyloid-Protein Aggregation

“…13, 14 As a common process that affects monoclonal-antibody (mAb) function, non-enzymatic deamidation also receives increasing attention in pharmaceutical and biotechnology industries. 13, 15 The half-lives of Asn and Gln deamidation vary extensively, from days to years, depending on the protein and many intrinsic and environmental factors. 8, 9 In general, Asn deamidates faster than Gln.…”

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS)

“…These modifications can include the oxidation of methionine and the deamidation of asparagine, among others. The separation and quantitation of peptides that have these modifications is of paramount importance in protein biotherapeutics because the modifications can contribute to a loss of stability or activity [1–3]. …”

“…This reaction is spontaneous and non-enzymatic, where asparagine residues undergo formation of a five-membered succinimide ring intermediate from an intramolecular attack, and subsequently hydrolyze under physiological conditions to form either aspartyl or isoaspartyl peptides, which can be found in both the D and L configurations (Figure 1). Deamidation occurs at a much faster rate (up to 70 times) when an unhindered amino acid residue such as glycine is on the C-terminal side of an asparagine in the primary sequence (XXX-Asn-Gly-XXX), but its rate is also affected by other conditions and characteristics such as temperature, pH, and protein structure [1, 4, 5, 8–12]. As deamidation changes the peptide/protein structure and conformation, it can significantly affect the function and stability of proteins.…”

“…For example, deamidation of an Asn-Gly site in hemoglobin alters its affinity for oxygen, while the same modification alters the proteolytic cleavage of human growth hormone (hGH) [13, 14]. Deamidation has been studied using different analytical techniques, such as isoelectric focusing, capillary electrophoresis, and a variety of LC-MS/MS techniques, but they all have limitations that make analyzing deamidation a challenge [1, 7]. The greatest challenge for mass spectrometric analysis of deamidated proteins is that there is only a 1 Dalton mass shift between the modified and native forms, which causes the deamidated species to overlap with the mass-to-charge ( m/z ) ratios of the 13 C isotopes of the unmodified species [15].…”