Monoclonal antibodies (mAbs) are complex glycoproteins that are usually produced using mammalian cells, resulting in complicated and heterogeneous post-translational modifications (PTMs). These are the results of a combination of cellular processing, chemical modification during purification, drug product filling, and/or storage. Among the various PTMs, N-terminal cyclization is one of the product quality attributes that may impact product quality, safety, and efficacy. Creative Biolabs now presents N-terminal cyclization analysis as part of the antibody PTM characterization services.
N-terminal cyclization, also called pyroglutamate formation (pyro-Glu), is one of the major N-terminal modifications of mAbs. proGlu variants are generated by the rearrangement of glutamine (Gln) and glutamic acid (Glu) at the N-terminus of mAb heavy chain (HC) and light chain (LC). This can be done chemically or enzymatically by glutaminyl cyclase. Nonenzymatic conversion of N-terminal Gln to pyroGlu can result in shifts toward lower molecular weights and lower isoelectric points. Chemically, cyclization of Gln to pyroGlu results in a loss of the N-terminal primary amine group, and therefore, these antibodies become slightly more acidic. Conversion of Gln to pyroGlu occurs primarily inside bioreactors, with little contribution from downstream purification and analytical processes.
Fig.1 N-terminal Cyclization of IgG antibodies. (Beck, 2013)
For Glu, on the other hand, this reaction can occur chemically after cell culture. Conversion of Glu to pyroGlu has been reported to occur on shelf storage and grow with time. Formulation factors, such as temperature, time, and pH can also impact the rate of non-enzymatic formation of pyro-Glu from Glu. Moreover, the formation of pyro-Glu can also be induced during analytical procedures. The conversion of Glu to pyroGlu results in a basic shift of antibody charges.
Due to its position away from the main complementary-determining regions (CDRs), few differences in antibody potency are noted when two HC N-terminal pyroGlu are compared with the same antibody with one pyroGlu and one Gln. Nevertheless, from a regulatory perspective, the N-terminal variants should still be considered in risk assessment and characterization studies to rule out impacts on product quality, safety, and efficacy. Their potential impact on antigen binding, immunogenicity, and Fc effector functions are required to be considered or evaluated experimentally.
As the conversion from Gln or Glu to pyroGlu results in charge variation of mAb products, this heterogeneity can be detected by charge-based methods such as cation-exchange (CEX) and capillary isoelectric focusing (cIEF). The conversion of Gln or Glu to pyroGlu gives a mass shift of -17 or -18 Da, respectively, compared to the unmodified peptide, and this can be assessed using peptide mapping and intact mass analysis by mass spectrometry (MS)-based methods.
Creative Biolabs has established a platform to analyze the structural variants of therapeutic mAb candidates. Our laboratories are equipped with a broad range of techniques that can satisfy various requirements. With considerable expertise in the analysis of PTMs, we can apply orthogonal analytical methods to identify a range of PTMs, including glycosylation, C-terminal lysine variant, N-terminal cyclization, deamidation & isomerization analysis, oxidation, glycation, and so on. We can fit different requirements of regulatory expectations and industry guidelines, such as the ICH Q6B guideline.
For more information, please contact us or send us an inquiry directly.
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