On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format.

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography-mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.

Development of a rapid reversed-phase liquid chromatographic method for total free thiol quantitation in protein therapeutics.

Free thiols, or unpaired cysteines, are important product quality attributes in the therapeutic proteins due to their potential impact on the protein structure, bioactivity and stability. While many free thiol quantitation methods were developed for specific therapeutic formats, an unmet need still exists for a multiproduct, high-throughput method for free thiol quantitation. In this study, a workflow was established that combines N-cyclohexylmaleimide (NcHM) derivatization and high-throughput reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) separation with superficially porous particle (SPP) column for quantitating total free thiols in monoclonal antibodies (mAbs), fragment antigen-binding (Fab), and bispecific antibodies (BsAbs). The NcHM derivatization increases the hydrophobicity of the free thiol variants and allows the further separation and quantitation with RP-UHPLC. A thorough evaluation of sample preparation, column selection, chromatographic condition and LC-MS peak identification was performed to optimize and characterize the method outputs. Method optimization resulted in a 42-minute total analysis time. Method qualification demonstrated suitable accuracy, precision, linearity, specificity and robustness. This high-throughput method is not only used for quantitation of total free thiols for both in-process testing and drug substance/drug product batch testing, but also for provide the positional distribution of the free thiols in the protein.

Development of a rapid RP-UHPLC-MS method for analysis of modifications in therapeutic monoclonal antibodies.

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (MAbs) that have high risk to safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic MAbs process development, thorough characterization and quantitative monitoring of CQAs requires a variety of analytical techniques. This paper describes the development of a rapid analytical method to assess modifications in MAbs, based on the analysis of subdomains with molecular weights of ∼25kDa. These subdomains were generated by digestion with a highly specific IdeS protease, followed by disulfide bond reduction. A reversed phase UHPLC-MS method was developed that provides efficient separation and identification of the subdomains (Fc, LC, and Fd) and related variants within 10min. Sample preparation and UHPLC instrument parameters were systematically evaluated. The methodology was applied to MAb stress panel characterization to capture the degradations induced by various stress conditions. Among the CQAs monitored by this method, Fc oxidation levels were compared with the values obtained by the more complicated and time-consuming peptide mapping method. The similar trends observed by the two methods demonstrated that the IdeS-UHPLC method is valuable as a higher throughput alternative to peptide mapping for monitoring modifications. In particular, a high-throughput methodology is preferred for analysis of the many samples associated with process development studies. Overall the method has been demonstrated as a fast, convenient and informative platform approach for analysis of therapeutic MAbs modifications including CQAs.

Characterization of free thiol variants of an IgG1 by reversed phase ultra high pressure liquid chromatography coupled with mass spectrometry.

RP-HPLC has been demonstrated as a powerful tool to study antibody free thiol and disulfide variants. Recently, the introduction of UHPLC columns with wide pore size (300Å) and small particle size (1.7µm) offered the opportunity to further improve the separation of such variants. This paper describes a systematic evaluation of stationary phases, operating parameters, and mobile phases for a UHPLC based method to separate free thiol variants of a recombinant monoclonal antibody (referred as mAb A), targeting high resolution, high throughput and improved recovery. Among the four different stationary phases evaluated, UHPLC diphenyl columns were found to provide the best separation. Using an optimized UHPLC method, free thiol variants of mAb A were separated in 5min. Importantly, the UHPLC method revealed minor variants that had coeluted in an HPLC based method, and the UHPLC method is also applicable as a platform method for characterization of other mAbs as well. Furthermore, an on-line UHPLC-MS method was developed to characterize the separated variants, and this method can streamline the characterization of fully assembled monoclonal and bispecific therapeutic antibodies.