Journal of Chromatography B (v.1032, #C)

Challenges in liquid chromatographic characterization of proteins by Balázs Bobály; Enikő Sipkó; Jenő Fekete (3-22).
Various liquid chromatographic techniques are considered standard analytical methods in proteins characterization. These methods provide essential information for drug approval, for biological and life sciences. On the other hand, there are some issues and challenges which have to be taken into account when analyzing these biopharmaceuticals. The aim of this review to summarize the most recent knowledge relating to the following topics: i) sample stability and complexity ii) adsorption problems: instrument inertness iii) adsorption problems: recovery from the stationary phase and iv) challenges in method development. This information is supposed to help practicing chromatographers in the emerging field of therapeutic protein chromatography.
Keywords: Sample stability; Protein chromatography; Adsorption; Method development; RPLC; IEX; SEC; HIC;

Challenges and opportunities of using liquid chromatography and mass spectrometry methods to develop complex vaccine antigens as pharmaceutical dosage forms by John M. Hickey; Neha Sahni; Ronald T. Toth; Ozan S. Kumru; Sangeeta B. Joshi; C. Russell Middaugh; David B. Volkin (23-38).
Liquid chromatographic methods, combined with mass spectrometry, offer exciting and important opportunities to better characterize complex vaccine antigens including recombinant proteins, virus-like particles, inactivated viruses, polysaccharides, and protein-polysaccharide conjugates. The current abilities and limitations of these physicochemical methods to complement traditional in vitro and in vivo vaccine potency assays are explored in this review through the use of illustrative case studies. Various applications of these state-of-the art techniques are illustrated that include the analysis of influenza vaccines (inactivated whole virus and recombinant hemagglutinin), virus-like particle vaccines (human papillomavirus and hepatitis B), and polysaccharide linked to protein carrier vaccines (pneumococcal). Examples of utilizing these analytical methods to characterize vaccine antigens in the presence of adjuvants, which are often included to boost immune responses as part of the final vaccine dosage form, are also presented. Some of the challenges of using chromatographic and LC–MS as physicochemical assays to routinely test complex vaccine antigens are also discussed.
Keywords: Chromatography; Mass spectrometry; Vaccine; Stability; Adjuvant; Formulation;

Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques by Tao Chen; Yan Chen; Cinzia Stella; Colin D. Medley; Jason A. Gruenhagen; Kelly Zhang (39-50).
Due to the inherent structure complexity and component heterogeneity of antibody drug conjugates (ADCs), separation technologies play a critical role in their characterization. In this review, we focus on chromatographic and electrophoretic approaches used to characterize ADCs with respect to drug-to-antibody ratio, drug distribution and conjugation sites, free small molecule drugs, charge variants, aggregates and fragments, etc. Chromatographic techniques including reversed-phase, ion exchange, size exclusion, hydrophobic interaction, two-dimensional liquid chromatography, and gas chromatography as well as capillary electrophoretic techniques including capillary electrophoresis sodium dodecyl sulfate, capillary zone electrophoresis and capillary isoelectric focusing are reviewed for their applications in the characterization of ADCs.
Keywords: Antibody drug conjugate (ADC); Critical quality attribute (CQA); Drug-to-antibody ratio (DAR); Drug distribution; Peptide mapping; Charge variants; Aggregations; Free drug analysis;

The development of analytical tools for the characterization of large biomolecules is an emerging and rapidly evolving area. This development activity is motivated largely by the current trend involving the increase in development and use of large biomolecules for therapeutic uses. Given the inherent complexity of these biomolecules, which arises from their sheer size and possibilities for chemical modification as well as changes over time (e.g., through modification in solution, aggregation), two-dimensional liquid chromatography (2D-LC) has attracted considerable interest as an analytical tool to address the challenges faced in characterizing these materials. The immediate potential benefits of 2D-LC over conventional one-dimensional liquid chromatography in this context include: (1) higher overall resolving power; (2) complementary information gained from two dimensions of separation in a single analysis; and (3) enabling indirect coupling of separation modes that are inherently incompatible with mass spectrometric (MS) detection (e.g., ion-exchange, because of high-salt eluents) to MS through a more compatible second dimension separation such as reversed-phase LC. In this review we summarize the work in this area, most of which has occurred in the past five years.Although the future is bright for further development in this area, some challenges have already been addressed through new 2D-LC methods. These include: (1) deep characterization of monoclonal antibodies to understand charge heterogeneity, glycosylation patterns, and other modifications; (2) characterization of antibody-drug conjugates to understand the extent and localization of small molecule conjugation; (3) detailed study of excipients in protein drug formulations; and (4) detection of host-cell proteins on biotherapeutic molecule preparations. We fully expect that in the near future we will see this list expanded, and that continued development will lead to methods with further improved performance metrics.
Keywords: Protein; Biopharmaceutical; Monoclonal antibody; Antibody-drug conjugate; Two-dimensional liquid chromatography; Mass spectrometry;

Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products by Rabah Gahoual; Alain Beck; Emmanuelle Leize-Wagner; Yannis-Nicolas François (61-78).
Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE–MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be dedicated to detail the current and state-of-the-art CE-based methods for the characterization of mAbs and related products.
Keywords: Monoclonal antibodies; Biosimilar; Fc-fusion protein; Antibody; Drug conjugate; Capillary gel electrophoresis; Capillary zone; Electrophoresis; Capillary isoelectric focusing; Mass spectrometry;

Over the past 15 years, monoclonal antibodies (mAbs) have emerged as the most successful class of therapeutics. Their specific structural and functional properties make them highly effective treatments for various diseases. Most therapeutic mAbs are based on chimeric, humanized or human G immunoglobulins (IgGs) selected from three isotypes (1, 2 and 4). IgGs are large and highly complex multimeric glycoproteins. They are constituted of a mixture of isoforms including macro and micro-variants that must be extensively characterized prior to their investigation as a drug candidate in clinical trials. The IgG backbone is also used to design more potent but also more complex biopharmaceuticals such as antibody-drug conjugates, bispecific antibodies, Fc-fusion proteins, and antibody mixtures to name a few. Mass spectrometric approaches in combination with electrophoretic and chromatographic separation methods play a central role in the analytical and structural multi-level (top, middle and bottom) characterization of these compounds. Importantly, techniques allowing the characterization of intact mAbs and related products under non-denaturing conditions are attracting increasing interest. Here, we review the current state of the art in native mass spectrometry and ion mobility methods for the characterization of mAbs and mAb-based products.
Keywords: Native mass spectrometry; Ion mobility mass spectrometry; Monoclonal antibody; Antibody-drug-conjugate; Bispecific antibody; Antibody/antigen complex;

This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris®). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC–MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC–MS in one single injection. It was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1, 3 and 5) and their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches.
Keywords: On-line comprehensive 2DLC; HICxRPLC-QTOF-MS; Quadrupole time-of-flight; Antibody-drug conjugate; ADCs; Odd DARs;

Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75 min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.
Keywords: On-line comprehensive 2D-LC; HICxRPLC-QTOF-MS; Antibody-drug conjugate; ADC; Brentuximab vedotin; Adcetris®;

Investigation of low recovery in the free drug assay for antibody drug conjugates by size exclusion—reversed phase two dimensional-liquid chromatography by Yi Li; Cinzia Stella; Laura Zheng; Charity Bechtel; Jason Gruenhagen; Fred Jacobson; Colin D Medley (112-118).
Antibody drug conjugates (ADCs) are complex therapeutic agents combining the selectivity of monoclonal antibodies and highly efficacious small molecule drugs to successfully eliminate tumor cells while limiting the general toxicity and side effects of the therapeutic to protect patient safety. One unique attribute critical to the safety of ADCs is the residual content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. Typically for quality control assays, quantifying the amount of the free drug is performed through precipitation of the protein species using an organic solvent and then assaying the amount of free drug left in the supernatant. During the validation of an assay of this type for a maleimide based linker drug, issues were experienced with low and variable recovery in the spiked samples of the drug substance and drug product. A two-dimensional heart-cutting method coupling Size Exclusion Chromatography (SEC) with Reverse Phase (RP) chromatography was utilized to explore possible mechanisms leading to the low recovery of the free linker drug. The results of the investigation indicated that the spiked linker drug reacts with residual reactive groups on the ADC; a conclusion which was confirmed by the observed increase of average Drug to Antibody Ratio (DAR) determined by Hydrophobic Interaction Chromatography (HIC). Finally, several approaches were evaluated to minimize the recovery loss. Capping the residual reactive groups on the ADC with maleimide containing reagents effectively mitigated the low recovery issue.
Keywords: Antibody drug conjugates; 2D-LC; Hydrophobic interaction chromatography;

Antibody-drug conjugates might be the magic bullets referred to by Paul Ehrlich over 100 years ago. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry to its limits. The use of multiple heart-cutting (mLC-LC) and comprehensive (LC × LC) multidimensional LC in combination with high resolution mass spectrometry for the characterization of the lysine conjugated antibody-drug conjugate ado-trastuzumab emtansine, commercialized as Kadcyla, is presented. By combining protein and peptide measurements, attributes such as drug loading, drug distribution and drug conjugation sites can be assessed in an elegant manner.
Keywords: Antibody-drug conjugate; Ado-trastuzumab emtansine; Two-dimensional liquid chromatography; Multiple heart-cutting; Comprehensive;

Importance of vial shape and type on the reproducibility of size exclusion chromatography measurement of monoclonal antibodies by Marta Rodriguez-Aller; Alessandra Cusumano; Beck Alain; Davy Guillarme; Szabolcs Fekete (131-138).
The goal of this work is to understand the phenomenon behind the poor injection repeatability often observed with protein biopharmaceuticals. All the measurements were carried out in size exclusion chromatography (SEC) mode, using fifteen commercially available therapeutic monoclonal antibodies (mAbs). First of all, we proved that the variation of peak areas between consecutive injections was much more critical with highly concentrated mAb samples (up to 80 mg/mL), while the SEC measurements of commercial mAbs having concentrations below 5 mg/mL were reliable. Second, we emphasized that the shapes, volumes and materials of the sample vial insert also contribute to the change of peak areas observed during consecutive injections. In this study, six different insert models were studied and the most critical were the ones possessing the narrowest conical shape at the bottom. Furthermore, the homogenization of samples (with pipette mixing rather than vortex) prior to analysis was of great interest and allows a significant improvement in injection repeatability. Finally, because the on-wall (on-insert) adsorption of mAbs reaches its equilibrium in around 50 min, it is advised to add a specified residence time prior to injection to achieve repeatable injection.
Keywords: Monoclonal antibody; Protein biopharmaceuticals; Size exclusion chromatography; Vial; Insert; Injection precision;

Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns by Marton Szigeti; Judit Bondar; Douglas Gjerde; Zsolt Keresztessy; Akos Szekrenyes; Andras Guttman (139-143).
N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10 min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.
Keywords: N-glycosylation; Enzyme immobilization; PNGase F; Capillary electrophoresis;

A simple LC/MRM–MS-based method to quantify free linker-payload in antibody-drug conjugate preparations by Wesley Zmolek; Stefanie Bañas; Robyn M. Barfield; David Rabuka; Penelope M. Drake (144-148).
Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM–MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10 nM with 370 pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step.
Keywords: Antibody-drug conjugate; ADC; Free drug; Multiple-reaction monitoring; MRM;

A new anti-human Fc method to capture and analyze ADCs for characterization of drug distribution and the drug-to-antibody ratio in serum from pre-clinical species by Mélissa Excoffier; Marie-Claire Janin-Bussat; Charlotte Beau-Larvor; Lysiane Troncy; Nathalie Corvaia; Alain Beck; Christine Klinguer-Hamour (149-154).
Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. They combine monoclonal antibody specificity for over-expressed tumor antigens and the high cytoxicity of small molecular drugs (SMDs) and can therefore selectively kill tumor cells while minimizing toxicity to normal cells. Nevertheless, the premature deconjugation of ADCs in the circulation may trigger off target toxicity in patients. The released free drug level must be low in circulation for an extended period of time as well as the de-conjugation rate to ensure an acceptable therapeutic window. As a result, the assessment of the stability of the linker between payload and mAb in the systemic circulation is of paramount importance before entering in clinical trial. Here we report a new universal method to immunocapture and analyze by LC–MS the stability and distribution of ADCs in sera from relevant preclinical species (mouse, rat and cynomolgus monkey). Furthermore we demonstrated that this workflow can be applied to both ADCs with cleavable and non cleavable linkers. Last but not least, the results obtained in cynomolgus serum using immunoprecipitation and LC–MS analysis were cross validated using an ELISA orthogonal method. As the ligand used for immunoprecipitation is targeting the Fc part of mAb (CaptureSelect™ Human IgG-Fc PK Biotin), this protocol can be applied to analyze the stability of virtually all ADCs in sera for preclinical studies without the need to prepare specific molecular tools.
Keywords: ADC; Stability in serum; DAR; De-conjugation; LC–MS analysis; ELISA;

Development and use of specific ELISA methods for quantifying the biological activity of bevacizumab, cetuximab and trastuzumab in stability studies by Inmaculada Suárez; Antonio Salmerón-García; José Cabeza; Luis Fermín Capitán-Vallvey; Natalia Navas (155-164).
Bevacizumab (BVZ), cetuximab (CTX) and trastuzumab (TTZ) are monoclonal antibodies (mAbs) used worldwide for the treatment of several widespread kinds of cancer. They are marketed as medicines under their respective tradenames: Avastin®, Erbitux® and Herceptin®. The aim of this research was to develop in-house specific enzyme-linked immunosorbent assays (ELISA) to assess the long-term stability of these three mabs. These assays assess the biological functionality of the mAbs by quantifying their biological activity. For this purpose, we developed an indirect ELISA procedure whereby the specific antigens against which the mAbs are directed are used as specific “capturing” antibodies on the ELISA plates. We therefore used vascular endothelial growth factor (VEGF) in the ELISA for BVZ; human epidermal growth factor receptor (hEGFR) in the ELISA for CTX and human receptor HER2 (hHER2) in the ELISA for TTZ. After the mAbs had attached to their antigen, we used an anti-human IgG (whole molecule) peroxidase-conjugate and o-phenylenediaminedihydrochloride substrate. The reaction was stopped using sulphuric acid and absorbance was recorded at a wavelength of 450 nm. The three ELISA methods were validated in terms of calibration models, range of the assay, limits of detection and quantitation, intra and interday precision and accuracy, and specificity by cross reactions. Forced degradation studies were also conducted on the medicines, providing useful information. Finally, the proposed ELISA were successfully used in a long-term stability study to quantify the remaining biological activity in medicines that had been opened and then stored under two different storage conditions, i.e. refrigerated at 4 °C and frozen at −20 °C. Results indicated that BVZ (Avastin®) is the most stable of the three in terms of its biological functionality.
Keywords: ELISA; Stability study; Monoclonal antibody based-medicines; Bevacizumab; Cetuximab; Trastuzumab;

Assessing analytical comparability of biosimilars: GCSF as a case study by Neh Nupur; Sumit Kumar Singh; Gunjan Narula; Anurag S. Rathore (165-171).
The biosimilar industry is witnessing an unprecedented growth with the newer therapeutics increasing in complexity over time. A key step towards development of a biosimilar is to establish analytical comparability with the innovator product, which would otherwise affect the safety/efficacy profile of the product. Choosing appropriate analytical tools that can fulfil this objective by qualitatively and/or quantitatively assessing the critical quality attributes (CQAs) of the product is highly critical for establishing equivalence. These CQAs cover the primary and higher order structures of the product, product related variants and impurities, as well as process related impurities, and host cell related impurities. In the present work, we use such an analytical platform for assessing comparability of five approved Granulocyte Colony Stimulating Factor (GCSF) biosimilars (Emgrast, Lupifil, Colstim, Neukine and Grafeel) to the innovator product, Neupogen®. The comparability studies involve assessing structural homogeneity, identity, secondary structure, and product related modifications. Physicochemical analytical tools include peptide mapping with mass determination, circular dichroism (CD) spectroscopy, reverse phase chromatography (RPC) and size exclusion chromatography (SEC) have been used in this exercise. Bioactivity assessment include comparison of relative potency through in vitro cell proliferation assays. The results from extensive analytical examination offer robust evidence of structural and biological similarity of the products under consideration with the pertinent innovator product. For the most part, the biosimilar drugs were found to be comparable to the innovator drug anomaly that was identified was that three of the biosimilars had a typical variant which was reported as an oxidized species in the literature. But, upon further investigation using RPC-FLD and ESI-MS we found that this is likely a conformational variant of the biotherapeutic been studied.
Keywords: Biosimilars; Analytical comparability; Peptide mapping; Mass spectroscopy; Granulocyte colony stimulating factor; GCSF;

Development of a rapid RP-UHPLC–MS method for analysis of modifications in therapeutic monoclonal antibodies by Bing Zhang; Justin Jeong; Braydon Burgess; Mansour Jazayri; Yun Tang; Yonghua Taylor Zhang (172-181).
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 ∼25 kDa. 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 10 min. 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.
Keywords: Monoclonal antibodies; MAbs; IdeS; UHPLC; Mass spectrometry; High resolution; Fc oxidation; CQAs; Peptide mapping; Process characterization and process validation (PC/PV) studies;

A comprehensive study to protein retention in hydrophobic interaction chromatography by Martyna Baca; Jelle De Vos; Gilles Bruylants; Kristin Bartik; Xiaodong Liu; Ken Cook; Sebastiaan Eeltink (182-188).
The effect of different kosmotropic/chaotropic salt systems on retention characteristics of intact proteins has been examined in hydrophobic interaction chromatography (HIC). The performance was assessed using different column chemistries, i.e., polyalkylamide, alkylamine incorporating hydrophobic moieties, and a butyl chemistry. Selectivity in HIC is mainly governed by the salt concentration and by the molal surface tension increment of the salt. Typically, a linear relationship between the natural logarithm of the retention factor and the salt concentration is obtained. Using a 250 mm long column packed with 5 μm polyalkylamide functionalized silica particles and applying a 30 min linear salt gradient, a peak capacity of 78 was achieved, allowing the baseline separation of seven intact proteins. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as calcium chloride (stabilizing the 3D conformation of α-lactalbumin) and isopropanol, on retention properties has been assessed. Results indicate that HIC retention is also governed by conformational in the proteins which affect the number of accessible hydrophobic moieties.
Keywords: Salting-out chromatography; Hofmeister; Hydrophobicity; Monoclonal antibodies; Conformational change;

Characterization and comparability of stress-induced oxidation and deamidation on vulnerable sites of etanercept products by Li-Juan Huang; Chia-Wang Chiang; Yu-Wen Lee; Tzu-Fan Wang; Ching-Chu Fong; Shu-Hui Chen (189-197).
An etanercept biosimilar, TuNEX®, was compared to the innovator drug, Enbrel®, for its reaction to stress-induced oxidation and deamidation, which may affect drug efficacy. A tryptic peptide map of both etanercept products was generated by liquid chromatography (LC) using mass spectrometry (MS) and ultraviolet (UV) spectrophotometry detection methods. The sequence of each modified or non-modified peptide peak was assigned based on accurate measurement of the mass of the protein and analysis utilizing tandem MS. Similar profiles of intrinsic oxidation on methionine (M) and deamidation on asparagine (N) were obtained for the two products, regardless of a two-amino acid (AA) residue variance in the heavy chain (Fc) between them. The level of oxidative stress exerted by tert-butyl hydroperoxide (tBHP), and alkaline stress exerted by a pH 10.4 solution, was examined using an LC-UV method. The results indicated that TuNEX® demonstrated a similar stress-induced modification profile compared to that of Enbrel®. For both products, oxidative stress increased the oxidation from an intrinsically low (0–6.9%) to moderate or high (42–100%) level for almost all M residues (M30, M174, M187, M223, M272, and M448); alkaline stress increased the deamidation level of N404 from a low (0.0 or 1.7%) to moderate (19–26%) level. Based the results of a cell-based bioactivity assay, TuNEX® also exhibited a similar level of bioactivity as Enbrel® in unstressed, oxidative-stressed, or alkaline-stressed conditions. The bioactivity of both products remained unaltered by oxidative stress but was reduced by alkali stress. In conclusion, our data indicated that TuNEX® exhibits a similar chemical stress profile as that of Enbrel® in terms of oxidation and deamidation as well as bioactivity.
Keywords: Etanercept; UPLC; MS; Oxidation; Deamidation; UV;

Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody by Kornél Viski; Zsolt Gengeliczki; Krisztián Lenkey; Katalin Baranyáné Ganzler (198-204).
Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.
Keywords: Monoclonal antibody; Glycation; Boronate affinity; Mass spectrometry;

Quantitative antibody-free LC–MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level by Daniel Wilffert; Riccardo Donzelli; Angela Asselman; Jos Hermans; Natalia Govorukhina; Nick H. ten Hacken; Wim J. Quax; Nico C. van de Merbel; Rainer Bischoff (205-210).
Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC–MS/MS method with a lower limit of quantitation (LLOQ) of ≈3 pM in induced sputum (174 pg/mL) and saliva (198 pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically 15N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC–MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.
Keywords: Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL); Saliva; Induced sputum; Antibody-free LC–MS/MS; Immobilized metal affinity (IMAC) enrichment; 15N-Metabolically labeled internal standard;

Quadrivalent human papillomavirus (HPV) vaccine is formulated of four types of non-infectious recombinant virus like particles (VLPs) that are structurally and immunologically similar to the corresponding infectious HPV virus types 6, 11, 16 and 18. With almost identical physical, chemical and structural properties of the four types of VLPs, ELISA remains the only approved in vitro potency testing assay. In this study, an alternative industry-friendly, stability- and potency-indicating assay protocol was developed and validated for the determination of HPV vaccine. Vacuum-driven immunoaffinity extraction (IAE) was employed using type-specific, conformation-dependent antibodies against each type of HPV VLPs. ELISA assay was employed to evaluate the ability of IAE columns to specifically separate each of the four types of VLPs from their quadrivalent mixture. Mean percentage recoveries of 76.76 ± 2.69, 69.12 ± 5.79, 84.86 ± 5.25 and 71.14 ± 4.50% were obtained for VLPs types 6, 11, 16 and 18, respectively with no significant interference in each case. Antigen content was then determined using SE-HPLC over a concentration range of 5.00–20.00 μg/mL (r > 0.998) for VLPs type 6, 11, 16 and 18, respectively. The SE-HPLC assay was found accurate and precise (RSD < 10.00%) with LOD ranging from 1.23–3.85 μg/mL. The assay protocol was found superior to conventional ELISA assay with respect to simplicity, total analysis time and cost. Good correlation between the results of analysis obtained using IAE—SE-HPLC and ELISA demonstrated the suitability of the suggested assay protocol for stability and potency assessment with a good potential for implementation for batch release. This approach should be applicable for quality assessment of other vaccine preparations based on VLPs.
Keywords: Immunoaffinity extraction; Size exclusion chromatography; Human papillomavirus vaccine; Virus like particles; Potency-indicating assay;

Characterization of N-glycosylation profiles from mammalian and insect cell derived chikungunya VLP by Catherine Lancaster; Pavlo Pristatsky; Van M. Hoang; Danilo R. Casimiro; Richard M. Schwartz; Richard Rustandi; Sha Ha (218-223).
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe arthralgia. The envelope of CHIKV is composed of 240 copies of two glycoproteins: E1 and E2. In this work, we have characterized the N-glycosylation patterns of CHIKV virus-like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells using hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FL) and mass spectrometry (MS) detection. While HEK293 derived CHIKV VLPs contain oligomannose, hybrid and complex glycans, VLPs derived from SfBasic predominantly contain oligomannose glycans. This strong host dependence of N-glycosylation pattern resembles other alphaviruses such as SINV. The VLPs from HEK293 and SfBasic, with significantly different N-glycosylation profiles, are valuable reagents enabling future in-depth correlation studies between immunogenicity and glycosylation. In addition, the characterization tools presented here allow one to monitor glycosylation during vaccine process development and ensure process consistency.
Keywords: Chikungunya; Vaccine; VLP; Glycosylation; HILIC;

Capillary electrophoresis techniques are widely used in the analytical biotechnology. Different electrophoretic techniques are very adequate tools to monitor size—and charge heterogenities of protein drugs.Method descriptions and development studies of capillary zone electrophoresis (CZE) have been described in literature. Most of them are performed based on the classical one-factor-at-time (OFAT) approach. In this study a very simple method development approach is described for capillary zone electrophoresis: a “two-phase-four-step” approach is introduced which allows a rapid, iterative method development process and can be a good platform for CZE method. In every step the current analytical target profile and an appropriate control strategy were established to monitor the current stage of development. A very good platform was established to investigate intact and digested protein samples.Commercially available monoclonal antibody was chosen as model protein for the method development study. The CZE method was qualificated after the development process and the results were presented. The analytical system stability was represented by the calculated RSD% value of area percentage and migration time of the selected peaks (<0.8% and <5%) during the intermediate precision investigation.
Keywords: Capillary zone electrophoresis; Method development; Therapeutic monoclonal antibody; Biotechnology; Charge variants;