The purpose of this work is the development and comparative assessment of two high resolution LCMS Attribute monitoring assays applied to monoclonal antibodies. Work presented here focuses on attribute monitoring at the IdeS subunit and peptide map levels ofanalysis for multiple product attributes.
Control and forced oxidized samples were prepared by exposing NIST mAb Reference material (RM8671) to 0, 0.003% and 0.01% of H2O2 respectively, at RT for 18 hr. For peptide mapping, the samples were subjected to rapid digestion following reduction and alkylation. LCMSE (DIA) data acquisition was used to generate qualitative and quantitative date on product attributes. IdeS digested subunit samples generated three ~25 kD subunits, resolved using an 8-min (total run time) RP method prior to MS analysis. Both peptide mapping and subunit mass analysis were carried out on a common UPLC/UV/QTof MS platform. Intrinsic modifications such as glycosylation, glycation and oxidation were monitored and compared using both workflows.
In this work, we have evaluated and compared two analytical workflows (peptide mapping and subunit mass) to demonstrate the capability of MS-based methods in monitoring several common biotherapeutic CQAs. A series of forced oxidized NIST RM8671 samples were used for attribute monitoring assay development, and to evaluate quantification of low abundance PTMs across a batch of samples.
For the subunit analysis, a short LC gradient was used to separate light chain, Fd and scFc fragments obtained from partial reduction with IdeS. The levels of glycosylation, glycation and oxidation were monitored across control and stressed sample sets and quantified using the ion counts from the charge deconvoluted spectra.
PTM characterization using conventional peptide mapping methods on NIST RM8671 was completed prior to the MAM assay development, with resulting attributes (defined by RT, m/z, and fragment ions) selected and saved to a library. The library, generated on the same analytical platform used for attribute monitoring is an integral part of the MAM analysis, and facilitates targeted attribute, quantification and trending. Eight Methionine sites (HC: M34, M87, M101, M255, M361, M431 and LC M4, M32) in the NIST mAb reference material were identified with high confidence fragmentation confirmation and used along with other negative control attributes to illustrate the analytical processing consistency and data trending across all samples. Additional key functions such as new peak detection, system suitability and limit checks were implemented to facilitate usability for process and quality monitoring.
In this study, the oxidation results obtained by subunit mass analysis were well correlated with the peptide attribute monitoring data. The quantification results demonstrated that both peptide mapping and subunit analyses have comparable repeatability and precision using this common analytical platform. Overall, we have demonstrated that these two approaches can potentially be employed ascomplementary assays for biotherapeutic PTM monitoring.