Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants

Abstract

Heterogeneity of therapeutic Monoclonal antibody (mAb) drugs are due to protein variants generated during the manufacturing process. These protein variants can be critical quality attributes (CQAs) depending on their potential impact on drug safety and/or efficacy. To identify CQAs and ensure the drug product qualities, a thorough characterization is required but challenging due to the complex structure of biotherapeutics. Past characterization studies for basic and acidic variants revealed that full characterizations were limited to the basic charge variants, while the quantitative measurements of acidic variants left gaps. Consequently, the characterization and quantitation of acidic variants are more challenging. A case study of a therapeutic mAb1 accounted for two-thirds of the enriched acidic variants in the initial characterization study. This led to additional investigations, closing the quantification gaps of mAb1 acidic variants. This work demonstrates that a well-designed study with the right choices of analytical methods can play a key role in characterization studies. Thus, the updated strategies for more complete antibody charge variant characterization are recommended.

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

“…Overall, these recent technical advances and applications reinforced the potential of this technology for reliable characterization of PTMs to assess CQAs and guarantee consistent quality in the manufacture of therapeutic mAbs throughout clinical and commercial development [39, 40].…”

Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control

“…A tryptic peptide mapping-based MAM does not report on charge isoform distribution or could underestimate glycated species, the commonly observed acidic species for a mAb. 19 However, according to authors from multiple companies 17 and Rogstad et al, 11 replacing a charge-based method, including IEC, is deemed acceptable due to MAM’s advantage providing site-specific charge-variant quantitation and better resolution of individual CQAs, compared to the overall charge distribution and co-eluted CQAs in a single IEC peak.…”

“…In addition, peptide-based MAM is great for the detection of single-site modifications (e.g., Fc glycans and DTLMISR oxidation), but is less suitable for, and often underestimates, a low-level modification at multiple sites. 19 Intact-level analysis might be needed to quantify such low-level multi-site modifications. Absolute quantitation of BsAb homodimers, 61 previously done in a development lab, can now be performed in a QC environment as needed, with QC compliant instruments and software.…”

“… Intact Level Conventional Method Attributes Contributing to the Product Variant Peptide-Based Standard MAM (Reduced, Trypsin*) Product Variant To monitor or control product variant For monitoring or control of the PQA? Charge ICIEF/IEC Deamidation Yes Oxidation Yes Hydroxylation Yes Fab glycan Yes Lys succinylation Yes Pyroglutamate Yes Glycation Yes (might underestimate 19 ) AGEs Yes O-glycan Yes C-terminal lysine Yes Isomerization Yes Succinmide Yes VHS extension Yes High Order Structure No LMWF Potential (depending on the fragment sequence, e.g., fragments that include tryptic cleavage sites may not be captured due to masking by the digested peptides, 11 and may not be as easy to quantitate ladder cleavages such as the common hinge-region fragmentation 20 ) Size R-CE-SDS NGHC Yes NR-CE-SDS LMWF Potential, see above SEC HMWF No Identity …”

In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry

“…7 Sialylation plays important roles in anti-inflammatory activity and antibody-dependent cellular cytotoxicity (ADCC). 8 Charge variants are often chosen as critical quality attributes of mAbs 9 and therefore shall be carefully characterized and closely monitored.…”