Therapeutic monoclonal antibodies (mAbs) are complex drug molecules with a high degree of heterogeneity, including charge variants, aggregates, fragments, and post-translational modifications (PTMs). PTMs can be introduced by chemical or enzymatic modifications during the production, storage, and in vivo circulation of mAbs. Creative Biolabs offers comprehensive characterization services of mAbs covering glycosylation, deamidation, oxidation, glycation, N-terminal cyclization, C-terminal lysine variant, etc. Here, we focus on the asparagine (Asn) deamidation and aspartate (Asp) isomerization as part of our PTM analysis and antibody degradation studies.
Deamidation of Asn transforms neutrally charged Asn residues to negatively charged Asp or iso-aspartate (iso-Asp) residues. This degradation event has been widely reported in recombinant mAbs in either the complementary-determining regions (CDRs) or in the constant regions during their manufacturing and storage processes. Deamidation of Asn in CDRs has been reported to affect antigen binding affinity and potency. Moreover, studies have also shown that changes in the charge distribution on the protein surface could alter mAb pharmacokinetics (PK) by affecting FcRn-IgG dissociation.
Fig.1 Antibody deamidation and isomerization. (Beck, 2013)
Native Asp itself can also undergo isomerization to form iso-Asp, which is another ubiquitous modification that can result in heterogeneity in mAbs. The sequences most sensitive to isomerization include Asp-Gly, Asp-Ser, and His-Asp. In this process, native Asp first converts to a cyclic succinimide intermediate and then either hydrolyzes back to Asp or isomerizes to iso-Asp. Studies have reported that Asp isomerization of CDR region of IgG antibodies results in reduced antibody potency and antigen binding.
Asn deamidation and Asp isomerization are two most frequently occurring degradation reactions during long-term storage of mAb products. Moreover, they can occur at different stages of the mAb production process. Product heterogeneity caused by uncontrolled degradation via deamidation and isomerization can complicate manufacturing consistency, thereby hampering long-term mAb functionality. Consequently, the identification of degradation-prone drug candidates is ideally done early in the drug development process to either adjust the manufacturing and formulation process accordingly or to re-engineer a problematic candidate to remove such hotspots.
Deamidation and isomerization are a quality-defining attribute of mAb therapeutics. Several analytical techniques can be used to detect either the degradation products (i.e. succinimide, Asp or iso-Asp), such as ion exchange chromatography (IEC) or capillary isoelectric focusing (cIEF). Separation of intact mAbs from their Asp isomerization products has been achieved using hydrophobic interaction chromatography (HIC). Besides, liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide maps are routinely used for the identification and quantification of deamidation and isomerization products.
Creative Biolabs now offers comprehensive antibody analytical services that cover detailed in vitro profiling of multiple mAbs to identify candidates that fulfill all desired functional criteria. Especially, we present our Asn deamidation and Asp isomerization analysis services to analyze mAb product heterogeneity under both normal and stressed conditions. Moreover, we can apply in silico prediction methods for the degradation propensity of both Asn and Asp residues on different sites of mAbs.
If you are interested in our service, please do not hesitate to contact us for more details.
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