High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats.In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.

(2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods, mAbs, 7:4, 732-742, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

The aim of this study was to characterize the product variants of a therapeutic T-cell bispecific humanized monoclonal antibody (TCB Mab, ∼200 kDa, asymmetric) and to develop an online cation-exchange chromatography native electrospray mass spectrometry method (CEC-UV-MS) for direct TCB Mab charge variant monitoring during bioprocess and formulation development. For the identification and functional evaluation of the diverse and complex TCB Mab charge variants, offline fractionation combined with comprehensive analytical testing was applied. The offline fractionation of abundant product variant peaks enabled identification of coeluting acid charge variants such as asparagine deamidation, primary and secondary Fab glycosylation (with and without sialic acid), and the presence of O-glycosylation in the G4S-linker region. Consequently, a new nonconsensus N-glycosylation motif (N-338-FG) in the heavy chain CDR region was discovered. Functional evaluation by cellbased potency testing demonstrated a clear and negative impact of both asparagine deamidations, whereas the O-glycosylation did not affect the TCB Mab biological activity. We established an online native CEC-UV-MS method, with an ammonium acetate buffer and pH gradient, to directly monitor the TCB Mab charge variants. All abundant chemical degradations and post-translational amino acid modifications already identified by offline fraction experiments and liquid chromatography mass spectrometry peptide mapping could also be monitored by the online CEC-UV-MS method. The herein reported online native CEC-UV-MS methodology represents a complementary or even alternative approach for multiattribute monitoring of biologics, offering multiple benefits, including increased throughput and reduced sample handling and intact protein information in the near-native state.

A new method for the quantification of benzoxazinone derivatives in extracts of wheat foliage and root samples using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation, and quantitative detection of a mixture of six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids (DIMBOA, DIBOA), lactams (HBOA, and HMBOA), benzoxazilinones (BOA, MBOA), and two synthetic methoxylated variations of DIBOA and HBOA, was achieved. The application of a novel, highly modified reversed-phase LC column, the dodecyl (C12) TMS end-capped Synergi MAX-RP, enhanced the on-line chromatographic separation through improvements to component resolution, analyte stability and peak shape and also to the column lifetime. The complete ESI-MS-MS precursor-product ion fragmentation pathways for the benzoxazinone derivatives are described for the first time and used to deduce a generic fragmentation pattern for the compound class. Characteristic transitions for the benzoxazinones were thus used in the developed analytical method enabling reliable quantification with simultaneous screening for other potentially present derivatives, while eliminating interferences from other coeluting contaminants from the complex plant extract matrix. Quantitative analysis was done in the multiple reaction monitoring mode, using two specific combinations of a precursor-product ion transitions for each compound. The ESI-MS-MS detection method offered improvements to the sensitivity and selectivity, as compared with previously applied LC methods, with detection limits down to 0.002-0.023 ng/microL. The developed method was demonstrated by analyzing foliages and roots of six different wheat cultivars using pressurized liquid extraction-solid-phase extraction cleanup-LC-ESI-MS-MS. The analytes were detected in the range of 0.7-207 microg/g of dry weight.