Identification of Hetero-aggregates in Antibody Co-formulations by Multi-dimensional Liquid Chromatography Coupled to Mass Spectrometry.

Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

Multi-Attribute Monitoring of Complex Erythropoetin Beta Glycosylation by GluC Liquid Chromatography-Mass Spectrometry Peptide Mapping.

Recombinant human erythropoetin (EPO) is an important biopharmaceutical mainly used for the treatment of anemia. It is highly heterogeneous because of common amino acid chemical degradations known to occur in protein therapeutics (e.g., oxidation and deamidation) and its complex glycosylation profile. Recently, multi-attribute monitoring (MAM), i.e., the quantification of multiple post-translational and chemical modifications in a single peptide mapping liquid chromatography-mass spectrometry (LC-MS)-based method, has received increased attention for the analysis of antibody-like biotherapeutic proteins. In this study, an MAM method for examination of residue-specific glycan profiles of EPO was established. The MAM method, by virtue of the increased sensitivity and selectivity provided with LC-MS, yielded additional site-specific information not afforded by the conventional quality control (QC) methods. Low abundant glycans as well as additional post-translational and chemical modifications could also be simultaneously detected by the MAM method. Our results demonstrate that desialylated N-oligosaccharides (DeNO) and N-acetylneuraminic acids (Neu5Ac) could be monitored by the developed MAM approach with data readout highly comparable to QC methods, while differences were observed for charge isoform distribution. In summary, the comparative data obtained demonstrate that MAM by LC-MS peptide mapping can, in principle, adequately replace selected QC methods and would add value to the in-process control and release testing strategy of EPO.

Application of Hydrogen/Deuterium Exchange-Mass Spectrometry to Biopharmaceutical Development Requirements: Improved Sensitivity to Detection of Conformational Changes.

The usefulness of the higher-order structure information provided by hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the protein therapeutic field is undisputed; however, its applicability as a method for critical quality and comparability assessment has until now not been demonstrated. Here we present results demonstrating for the first time the applicability of the HDX-MS technique to monitor structural changes due to methionine oxidation at sensitivity levels realistic to the requirements of biopharmaceutical research and development. For the analyzed heavy chain marker peptides deuterium uptake differences due to oxidation at the conserved methionine in position 254 were significantly verifiable at the lowest increase (1%) through spiked oxidized IgG1.

Mapping the Interactions of Selective Biochemical Probes of Antibody Conformation by Hydrogen-Deuterium Exchange Mass Spectrometry.

Protein-based pharmaceuticals represent the fastest growing group of drugs in development in the pharmaceutical industry. One of the major challenges in the discovery, development, and distribution of biopharmaceuticals is the assessment of changes in their higher-order structure due to chemical modification. Here, we investigated the interactions of three different biochemical probes (Fab s) generated to detect conformational changes in a therapeutic IgG1 antibody (mAbX) by local hydrogen-deuterium exchange mass spectrometry (HDX-MS). We show that two of the probes target the Fc part of the antibody, whereas the third probe binds to the hinge region. Through HDX-ETD, we could distinguish specific binding patterns of the Fc -binding probes on mAbX at the amino-acid level. Preliminary surface plasmon resonance (SPR) experiments showed that these domain-selective Fab probes are sensitive to conformational changes in distinct regions of a full-length therapeutic antibody upon oxidation.

Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals.

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Quantifying methionine sulfoxide in therapeutic protein formulation excipients as sensitive oxidation marker.

Methionine is a common excipient used in therapeutic protein liquid formulations as stabilizer and antioxidant. The oxidation of methionine to methionine sulfoxide can be regarded as a sensitive marker of oxidative stress for drug product storage conditions. In this study, a sensitive HPLC method for the quantification of methionine sulfoxide in formulated protein product was developed and qualified according to regulatory requirements using a SIELC® Primesep 100 column with UV detection. The separation involves a mixed-mode mechanism including reversed phase and cationic exchange modalities. The operating range of the method was established between 1 µM and 35 µM of methionine sulfoxide. In this testing range, the method was shown to be linear (R2 > 0.99), accurate (Recovery 92.9 - 103.6%, average recovery = 99.8 ± 1.4%) and precise (intermediate precision at LoQ, CV = 2.9%). The developed test system was successfully applied to study the effects of temperature and storage conditions on methionine sulfoxide formation in complex therapeutic antibody formulations.

Multiattribute Monitoring of Antibody Charge Variants by Cation-Exchange Chromatography Coupled to Native Mass Spectrometry.

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 cell-based 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.

The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange.

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

Effects of sialic acid linkage on antibody-fragment crystallizable receptor binding and antibody dependent cytotoxicity depend on levels of fucosylation/bisecting.

Aim: Fragment crystallizable (Fc) glycosylation of immunoglobulin G-type monoclonal antibodies applied to therapeutic applications is regarded a critical quality attribute and can influence bioactivity, pharmacokinetics and/or immunogenicity/safety. Investigating the impact of certain Fc N-glycans is therefore of importance to assess its criticality for a therapeutic product. This has been done for N-glycan types like fucosylation, galactosylation or sialylation. There were contradictory results reported for functionality especially with regard to sialylation. Material & methods: We elucidated the effect of terminal sialic acid residues on Fcγ receptor binding and antibody dependent cytotoxicity activity of two immunoglobulin G1 antibodies with different levels of fucosylation/bi-secting. Conclusion: We found the impact to be specific to the sialylation linkage type, in other words, α2,3- versus α2,6-linked sialic acid attached to the terminal galactose residues.

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

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.

Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry.

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.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods.

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.

Functional assessment of antibody oxidation by native mass spectrometry.

Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis. In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.

Development of a liquid chromatography-electrospray-tandem mass spectrometry method for the quantitative determination of benzoxazinone derivatives in plants.

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.