Monoclonal antibody (mAb)-based therapeutics now represent a new and rapidly growing class of drugs to treat a number of diseases. The analytical characterization of a new biotherapeutic protein covers a wide range of aspects, among which the comprehensive characterization of post-translational modifications (PTMs) is of utmost importance. As an expert in therapeutic antibody development and characterization, Creative Biolabs employs PTM analysis as part of our in-depth antibody structure analysis services.
PTMs can be classified into two major classes, enzymatic and chemical modifications. Enzymatic modifications are defined as enzyme-catalyzed processing of proteins after translation by different kinases, phosphatases, proteases, transferases, ligases, etc. Most common PTMs in this class include glycosylation, disulfide bond formation, and proteolytic cleavage of the protein backbone. Chemical modifications are often generated during downstream processing, formulation, and storage. Common chemical modifications include oxidation, deamidation, glycation, and pyroglutamate formation. The diversity of PTMs can affect the biological activity, half-life, and immunogenicity of mAb products, and product heterogeneity may result in safety problems. This necessitates that PTMs of antibody products must be comprehensively characterized, controlled, and monitored during the therapeutic mAb development process.
Fig.1 Different types of PTM modifications and alterations. (Higel, 2016)
Glycosylation is a major PTM affecting protein folding, conformation, stability, solubility, localization, and activity. Most therapeutic mAbs have an N-glycosylation site on Fc fragment, which is known to affect their effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The diversity of glycans has made them major contributors to mAb heterogeneity. Creative Biolabs offers glycosylation analysis of mAb products as well as Fc glycoengineering services to optimize the performance of your antibody candidates.
C-terminal Lys variants are clipped modification found at heavy chain C-terminus of mAbs produced in mammalian cell cultures. Because Lys residue is positively charged, its loss results in a decrease in positive charge, leading to charge heterogeneity of mAb products. Therefore, it is a quality parameter required to be characterized.
N-terminal cyclization (pyro-Glu) variants are generated by the rearrangement of Gln or Glu at the N-terminus of mAbs, which can occur spontaneously or be catalyzed enzymatically by glutaminyl cyclase. The conversion from Gln to pyroGlu renders mAbs more acidic and the conversion from Glu to pyroGlu gives a basic shift. Thus, the N-terminal cyclization increases charge heterogeneity of mAb products, which can be detected using charge-based techniques.
Deamidation of asparagines (Asp) is commonly observed and has an important role in regulating the heterogeneity and stability of recombinant mAbs. Besides, native Asp can undergo isomerization to form isoaspartic acid (iso-Asp), which is another ubiquitous modification that can result in heterogeneity in mAbs.
Antibody residues, such as methionine, cysteine, are tyrosine, are susceptible to oxidation. This process can occur in different stages of mAb development, including production, purification, formulation, and storage, thereby altering their physical and biological properties and affecting their potency and stability.
Glycation refers to the non-enzymatic addition of a monosaccharide to an amino acid residue via the Maillard reaction. The main source of this PTM is the reducing sugars in cell media (e.g., glucose, galactose). It introduces heterogeneity into the sample, particularly in regards to the charge profile.
Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. Incomplete formation of disulfide bonds results in the formation of different cysteine-related variants, including free sulfhydryls, thioether formation, cysteinylation, and trisulfide bond, etc.
Creative Biolabs offers PTM analysis by using the mass spectrometry (MS)-based methods, which has become the dominant tools in mAb structure characterization for years. MS-based approaches can provide high reproducibility, sensitivity, specificity, precision, accuracy, and multiplexing. They can identify different types of PTMs, including N-terminal and C-terminal variants, oxidation, amidation, deamidation, disulfide bonds, etc. We can offer bottom-up, top-down, and middle-up approaches to suit the diversified requirements. Moreover, we apply our knowledge to design strategic analytical programs based on your product’s specific structural characteristics. We also apply a range of chromatographic techniques to detect and identify different PTMs.
Some of these chemical or enzymatic modifications have resulted in the charge heterogeneity of mAb products, including C-terminal lysine cleavage, N-terminal cyclization, deamidation, oxidation, glycation, etc. Therefore, Creative Biolabs also offers an antibody charge variant analysis package to isolate and analyze different charge isoforms.
Besides, Creative Biolabs also offers antibody forced degradation studies to characterize therapeutic mAb stability by exposing it to sub-optimal temperatures, mechanical stresses, light, pH, freeze/thaw cycles, and so forth. If you are interested in our services, please do not hesitate to contact us for more details.
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