A rapid method for relative quantification ofN-glycans from a therapeutic monoclonal antibody during trastuzumab biosimilar development

Abstract

Glycosylation is a common post-translational modification and critical quality attribute that can modulate the efficacy of therapeutic proteins. In the production of monoclonal antibodies (mAbs), quantifying the glycoform profile is a vital characterization step. Traditional glycan analysis is time consuming and involves steps at extreme temperature or pH, which may alter glycans. Here, we describe a rapid method for glycan analysis in which glycans are released from mAb samples that are bound to protein A columns. Since host cell proteins, which may also contain glycans, were already removed, this step enables analysis of cell culture products. Glycans released from the mAb samples are then derivatized with InstantPC™ labeling agent and analyzed by HILIC-FLD-MS. To illustrate the method, the glycan profiles of six trastuzumab (Herceptin®) antibody lots and four biosimilar developmental lots were analyzed. The results derived from our novel method, which takes less than 90 min, are compared with those from a typical glycan preparation approach.

Minor N-Glycan Mapping of Monoclonal Antibody Therapeutics Using Middle-Down NMR Spectroscopy

“…The p value of 1.4 × 10 –4 demonstrated the significant difference in high mannose level between the two mAbs. Within the data precision, the NMR results of (M4–M9)% were consistent with the published results using orthogonal methods, 8.0–9.2% and 1.3–5.2% for adalimumab , and trastuzumab, − respectively (Table ).…”

“…The difference in galactosylation between the two mAbs was significant as p values were well below 0.05 (Table ). The Gal 1–3 % of adalimumab and trastuzumab recalculated from literature results were in the range of 7–9% and 16–17%, respectively; and the recalculated Gal 1–6 % of adalimumab and trastuzumab were in the range of 13–19% and 33–38%, respectively, when glycan mapping methods were applied. − In summary, all the NMR measured branch specific Gal 1–3/6 % of the two mAbs were in good agreement with the results of orthogonal measurements.…”

“…The previously published studies using glycan mapping methods have identified total aFuc% to be in the range of 6.0–11% and 5.8–12% in adalimumab and trastuzumab, respectively (Table ). − The total aFuc% measured using NMR was in the upper range of the literature values, probably due to the greater flexibility of afucosylated GlcNAc2­(aF), leading to slightly higher values in eq .…”

Establishment of a highly precise multi-attribute method for the characterization and quality control of therapeutic monoclonal antibodies

“…The average relative peak area percentages of 17 glycopeptides from all experiments are shown in Figure 7 . The average relative peak area of glycopeptides having three major glycans, A2G0F, A2G1F and A2G2F, was 39.46% (CV, 0.26%), 32.87% (CV, 0.41%) and 5.01% (CV, 1.12%), respectively, which agrees with a previous report [ 15 ]. Minor components, such as M6 and A1G1M5 (hybrid-type glycan), were also detected successfully.…”

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

“…Two commercial kits called RapiFluor-MS (RFMS) and InstantPC have also been developed to reduce the hydrolysis of glycosylamine. These two kits involve fast PNGase F digestion procedures and rapid N-glycan derivatization by activating carbamate chemistry in less than 10 min [64,70]. Advantages of these tools include improved ionization from tertiary amine groups and enhanced fluorescent intensity from fluorophores.…”