Publications by authors named "Davy Guillarme"

242 Publications

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies.

J Chromatogr A 2021 Aug 26;1655:462499. Epub 2021 Aug 26.

Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland. Electronic address:

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.
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http://dx.doi.org/10.1016/j.chroma.2021.462499DOI Listing
August 2021

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of .

Front Chem 2021 12;9:664489. Epub 2021 Aug 12.

School of Pharmaceutical Sciences, University of Geneva, CMU, Geneva, Switzerland.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm . The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as ()-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of .
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http://dx.doi.org/10.3389/fchem.2021.664489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397479PMC
August 2021

Inter-laboratory study to evaluate the performance of automated online characterization of antibody charge variants by multi-dimensional LC-MS/MS.

Talanta 2021 Nov 18;234:122628. Epub 2021 Jun 18.

Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA. Electronic address:

An international study was conducted to evaluate the performance and reliability of an online multi-dimensional (mD)-LC-MS/MS approach for the characterization of antibody charge variants. The characterization of antibody charge variants is traditionally performed by time-consuming, offline isolation of charge variant fractions by ion exchange chromatography (IEC) that are subsequently subjected individually to LC-MS/MS peptide mapping. This newly developed mD-LC-MS/MS approach enables automated and rapid characterization of charge variants using much lower sample requirements. This online workflow includes sample reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis within a single, fully-automated procedure. The benefits of using online mD-LC-MS/MS for variant characterization include fewer handling steps, a more than 10-fold reduction in required sample amount, reduced sample hold time as well as a shortening of the overall turnaround time from weeks to few days compared to standard offline procedures. In this site-to-site comparison study, we evaluated the online peptide mapping data collected from charge variants of trastuzumab (Herceptin®) across three international laboratories. The purpose of this study was to compare the overall performance of the online mD-LC-MS/MS approach for antibody charge variant characterization, with all participating sites employing different mD-LC-MS/MS setups (e.g., instrument vendors, modules, columns, CDS software). The high sequence coverage (95%-97%) obtained in each laboratory, enabled a reproducible generation of tryptic peptides and the comparison of values of the charge variants. Results obtained at all three participating sites were in good agreement, highlighting the reliability and performance of this approach, and correspond with data gained by the standard offline procedure. Overall, our results underscore of the benefit mD-LC-MS/MS technology for therapeutic antibody characterization, confirming its potential to become an important tool in the toolbox of protein characterization scientists.
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http://dx.doi.org/10.1016/j.talanta.2021.122628DOI Listing
November 2021

Ion mobility-high resolution mass spectrometry in doping control analysis. Part II: Comparison of acquisition modes with and without ion mobility.

Anal Chim Acta 2021 Aug 7;1175:338739. Epub 2021 Jun 7.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits. The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20-40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied. Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.
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http://dx.doi.org/10.1016/j.aca.2021.338739DOI Listing
August 2021

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

J Pharm Biomed Anal 2021 Sep 12;203:114206. Epub 2021 Jun 12.

Analytical Research and Development, MRL, Merck & Co, Inc., 126 E. Lincoln Ave, Rahway, NJ 07065, United States.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.
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http://dx.doi.org/10.1016/j.jpba.2021.114206DOI Listing
September 2021

Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography.

J Chromatogr A 2021 Aug 6;1651:462320. Epub 2021 Jun 6.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to "non-proportional" behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved - ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use.
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http://dx.doi.org/10.1016/j.chroma.2021.462320DOI Listing
August 2021

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis.

Pharmaceuticals (Basel) 2021 May 24;14(6). Epub 2021 May 24.

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Université de Strasbourg, CNRS, 67087 Strasbourg, France.

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.
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http://dx.doi.org/10.3390/ph14060498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225019PMC
May 2021

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

J Chromatogr A 2021 Aug 18;1650:462258. Epub 2021 May 18.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.
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http://dx.doi.org/10.1016/j.chroma.2021.462258DOI Listing
August 2021

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS.

Methods Mol Biol 2021 ;2271:85-95

School of Pharmaceutical Sciences, University of Geneva, CMU, Geneva, Switzerland.

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 μm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS.
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http://dx.doi.org/10.1007/978-1-0716-1241-5_6DOI Listing
June 2021

Fast Afucosylation Profiling of Glycoengineered Antibody Subunits by Middle-Up Mass Spectrometry.

Methods Mol Biol 2021 ;2271:73-83

Pierre Fabre Laboratories, IRPF-Centre d'Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France.

Middle-up LC-MS antibody characterization workflows using reduction or IdeS digestion for a focused assessment of N-glycan profiling of three representative glycoengineered monoclonal antibodies (mAbs), namely, obinutuzumab (GlycomAb technology, Glycart/Roche), benralizumab (Potelligent Technology, BioWa, Kyowa Kirin) and mAb B (kifunensine) and compared to mAb A, produced in a common CHO cell line. In addition, EndoS or EndoS2 enzyme are used for quantitative determination of Fc-glycan core afucosylation and high mannose for these antibodies, as requested by health authorities for Fc-competent therapeutics mAbs critical quality attributes (CQAs).
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http://dx.doi.org/10.1007/978-1-0716-1241-5_5DOI Listing
June 2021

Aptamer-based immunoaffinity LC-MS using an ultra-short column for rapid attomole level quantitation of intact mAbs.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Apr 5;1173:122694. Epub 2021 Apr 5.

Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, United States.

Quantification of proteins in biofluids has largely involved either traditional ligand binding assays or "bottom-up" mass spectrometry. Recently, top-down mass spectrometry using reversed-phase liquid chromatography (RPLC) paired with high-resolution mass spectrometry (HRMS) has emerged as a promising technique, due to the potential of better identification of post-translational modifications (PTMs), lack of downstream interferences, and less time-consuming sample preparation and analysis times. However, it can be difficult with this approach to robustly obtain high-fidelity MS data, especially when pushing for low limits of detection. To address these issues, we developed a chromatographic device with an optimized form factor and stationary phase to improve protein recovery, while reducing run times. We have observed that by using this device, it is possible to achieve attomole quantitation of mAbs without the addition of carrier proteins and with over three-fold higher throughput than columns employed in previous studies. Moreover, we have devised a novel affinity capture method, based on repurposing a unique aptamer ligand that can give 93% recovery of mAb using only a 2 h incubation. When hyphenated together, these two technologies greatly improve the ability to analyze proteins in complex matrices.
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http://dx.doi.org/10.1016/j.jchromb.2021.122694DOI Listing
April 2021

Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography - Mass spectrometry.

Anal Chim Acta 2021 Apr 24;1156:338347. Epub 2021 Feb 24.

Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA. After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals.
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http://dx.doi.org/10.1016/j.aca.2021.338347DOI Listing
April 2021

Expanding the range of sub/supercritical fluid chromatography: Advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis.

J Chromatogr A 2021 Apr 9;1642:462048. Epub 2021 Mar 9.

School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, 1211 Geneva 4, Switzerland. Electronic address:

The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level. Furthermore, the use of reduced percentages of MSA, such as 8 mM, yielded similar results to those observed with 15 mM of MSA. The optimized UHPSFC method was, then, used to compare the performance of UHPSFC against RP-UHPLC for peptides with different pI and with increasing peptide chain length. UHPSFC was found to give a slightly better separation of the peptides according to their pI values, in few cases orthogonal to that observed in UHPLC. On the other hand, UHPSFC produced a much better separation of peptides with an increased amino acidic chain compared to UHPLC. Subsequently, UHPSFC-MS was systematically compared to UHPLC-MS using a set of linear and cyclic peptides commercially available. The optimized UHPSFC method was able to generate at least similar, and in some cases even better performance to UHPLC with the advantage of providing complementary information to that given by UHPLC analysis. Finally, the analytical UHPSFC method was transferred to a semipreparative scale using a proprietary cyclic peptide, demonstrating excellent purity and high yield in less than 15 min.
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http://dx.doi.org/10.1016/j.chroma.2021.462048DOI Listing
April 2021

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies.

J Chromatogr A 2021 Apr 9;1642:462050. Epub 2021 Mar 9.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.
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http://dx.doi.org/10.1016/j.chroma.2021.462050DOI Listing
April 2021

Ion mobility-high resolution mass spectrometry in anti-doping analysis. Part I: Implementation of a screening method with the assessment of a library of substances prohibited in sports.

Anal Chim Acta 2021 Apr 31;1152:338257. Epub 2021 Jan 31.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis. In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, CCS values, and m/z ratios was constructed. Urine samples were analyzed using either a simple "dilute and shoot" procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and CCS values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.
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http://dx.doi.org/10.1016/j.aca.2021.338257DOI Listing
April 2021

Algorithms to optimize multi-column chromatographic separations of proteins.

J Chromatogr A 2021 Jan 23;1637:461838. Epub 2020 Dec 23.

Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

The goal of this work was to provide a technical solution for the automated optimization of multi-column systems for protein separation and fractionation. Both algorithm and a software that can be downloaded are provided. In this algorithm, the length and order of the individual column segments can be considered. Various solutions are provided by the algorithm, including i) to obtain uniform peak distribution, ii) to park the different species at the inlet of the individual column segments, and iii) to elute all species as a single peak. Two representative examples are presented, showing the possibility to obtain uniform selectivity between monoclonal antibody (mAb) sub-units, and the on-column fractioning of intact mAbs.
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http://dx.doi.org/10.1016/j.chroma.2020.461838DOI Listing
January 2021

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches.

Analyst 2021 Feb 7;146(3):747-769. Epub 2021 Jan 7.

Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Accelerated development of new therapeutics in an increasingly competitive landscape requires the use of high throughput analytical platforms. In addition, the complexity of novel biotherapeutic formats (e.g. fusion proteins, protein-polymer conjugates, co-formulations, etc.) reinforces the need to improve the selectivity and resolution of conventional one-dimensional (1D) liquid chromatography (LC). Liquid chromatography-mass spectrometry (LC-MS)-based technologies such as native LC-MS for intact mass analysis or peptide mapping (also called bottom-up approach)-based multi-attribute methods (MAM) have already demonstrated their potential to complement the conventional analytical toolbox for monoclonal antibody (mAb) characterization. Two-dimensional liquid-chromatography (2D-LC-MS) methods have emerged in the last ten years as promising approaches to address the increasing analytical challenges faced with novel antibody formats. However, off-line sample preparation procedures are still required for conventional 1D and 2D-LC-MS methods for the in-depth variant characterization at the peptide level. Multi-dimensional LC-MS (mD-LC-MS) combine sample preparation and multi-level (i.e. intact, reduced, middle-up and peptide) analysis within the same chromatographic set-up. This review presents an overview of the benefits and limitations of mD-LC-MS approaches in comparison to conventional chromatographic methods (i.e. 1D-LC-UV methods at intact protein level and 1D-LC-MS methods at peptide level). The current analytical trends in antibody characterization by mD-LC-MS approaches, beyond the 2D-LC-MS workhorse, are also reviewed, and our vision on a more integrated multi-level mD-LC-MS characterization platform is shared.
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http://dx.doi.org/10.1039/d0an01963aDOI Listing
February 2021

Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept.

Anal Chem 2021 01 17;93(3):1277-1284. Epub 2020 Dec 17.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes. Moreover, long columns-in theory-just broaden the peaks of large solutes since a great part of the column only acts as void (extra) volume. Such a theory suggests that using very short columns should result in comparable separation quality versus using long columns and make it possible to perform faster (high-throughput) analyses. Therefore, the elution behavior of various therapeutic monoclonal antibodies and their fragments (25-150 kDa) has been investigated using modern instrumentation and column formats. The possibilities offered by narrow-bore columns packed with state-of-the-art 2.7 μm superficially porous particles with 5, 50, 100, and 150 mm lengths have been compared. In particular, the impact of gradient steepness and column length on separation efficiency was evaluated. Using 5 mm × 2.1 mm columns, it has become possible to separate antibody fragments and antibody-drug conjugate species in less than 30 s. Such fast methods can be very useful for high-throughput screening purposes in biopharmaceutical industries.
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http://dx.doi.org/10.1021/acs.analchem.0c04082DOI Listing
January 2021

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography.

Anal Chem 2021 01 11;93(3):1285-1293. Epub 2020 Dec 11.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland.

In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths. It was found that on a low dispersion ultrahigh-pressure liquid chromatography instrument, columns between 10 and 20 mm were most effective when made with 2.1 mm i.d. tubing. However, with the same LC instrument, 3 mm i.d. columns as short as ∼5 to 10 mm could be effectively used. In both cases, it has been found to be best to keep injection volumes below 0.6 μL, which presents a potential limit to further decreasing column length, given the current capabilities of autosampler instrumentation. The additional volume of the column hardware outside of the packed bed (extra-bed volume) of very small columns is also a limiting factor to decrease the column length. For columns shorter than 10 mm, columns' extra-bed volume was seen to make considerable contributions to band broadening. However, the use of ultra-short columns seemed to be a very useful approach for RPLC of large proteins (>25 kDa) and could also work well for ∼12 kDa as the lowest limit of molecular mass. In summary, a renewed interest in the use of ultra-short columns is warranted, and additional method development will be to the benefit of the biopharmaceutical industry as there is an ever-increasing demand for faster, yet accurate assays (, high-throughput screening) of proteins.
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http://dx.doi.org/10.1021/acs.analchem.0c01720DOI Listing
January 2021

Negative gradient slope methods to improve the separation of closely eluting proteins.

J Chromatogr A 2021 Jan 23;1635:461743. Epub 2020 Nov 23.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples. This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted. First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an "on-off" type elution behavior. Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.
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http://dx.doi.org/10.1016/j.chroma.2020.461743DOI Listing
January 2021

Supercritical fluid chromatography - Mass spectrometry in metabolomics: Past, present, and future perspectives.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Dec 17;1161:122444. Epub 2020 Nov 17.

VU Amsterdam, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Division of BioAnalytical Chemistry, Amsterdam, the Netherlands; Center for Analytical Sciences Amsterdam, Amsterdam, the Netherlands. Electronic address:

Metabolomics, which consists of the comprehensive analysis of metabolites within a biological system, has been playing a growing role in the implementation of personalized medicine in modern healthcare. A wide range of analytical approaches are used in metabolomics, notably mass spectrometry (MS) combined to liquid chromatography (LC), gas chromatography (GC), or capillary electrophoresis (CE). However, none of these methods enable a comprehensive analysis of the metabolome, due to its extreme complexity and the large differences in physico-chemical properties between metabolite classes. In this context, supercritical fluid chromatography (SFC) represents a promising alternative approach to improve the metabolome coverage, while further increasing the analysis throughput. SFC, which uses supercritical CO as mobile phase, leads to numerous advantages such as improved kinetic performance and lower environmental impact. This chromatographic technique has gained a significant interest since the introduction of advanced instrumentation, together with the introduction of dedicated interfaces for hyphenating SFC to MS. Moreover, new developments in SFC column chemistry (including sub-2 µm particles), as well as the use of large amounts of organic modifiers and additives in the CO-based mobile phase, significantly extended the application range of SFC, enabling the simultaneous analysis of a large diversity of metabolites. Over the last years, several applications have been reported in metabolomics using SFC-MS - from lipophilic compounds, such as steroids and other lipids, to highly polar compounds, such as carbohydrates, amino acids, or nucleosides. With all these advantages, SFC-MS is promised to a bright future in the field of metabolomics.
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http://dx.doi.org/10.1016/j.jchromb.2020.122444DOI Listing
December 2020

Evaluation of Different Tandem MS Acquisition Modes to Support Metabolite Annotation in Human Plasma Using Ultra High-Performance Liquid Chromatography High-Resolution Mass Spectrometry for Untargeted Metabolomics.

Metabolites 2020 Nov 15;10(11). Epub 2020 Nov 15.

School of Pharmaceutical Sciences, University of Geneva, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland.

Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.
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http://dx.doi.org/10.3390/metabo10110464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697060PMC
November 2020

Investigating the use of unconventional temperatures in supercritical fluid chromatography.

Anal Chim Acta 2020 Oct 18;1134:84-95. Epub 2020 Aug 18.

School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

The use of unorthodox temperatures, ranging from -5 °C up to 80 °C, have been thoroughly investigated in supercritical fluid chromatography. To this purpose, an initial evaluation of the kinetic and thermodynamic performance has been made with a set of 4 analytes eluting at different percentages of organic co-solvent in the mobile phase (3%-10% - 45%-80%). The van Deemter plots have demonstrated how, at low organic modifier presence, the use of low temperatures did not necessarily translate into worse performance, while high temperatures could pose more issues due to the poor handling of the super/subcritical mobile phase by the chromatographic system. With important percentages of co-solvent, however, high temperatures were fundamental in ensuring better profiles of the van Deemter plots, compared to low temperatures. Pressure plots have demonstrated that gradients reaching elevated percentages of organic modifiers can also be used on stationary phases packed with sub 2 μm silica particles if high temperatures are employed. The thermodynamic evaluation, made via the analysis of van't Hoff plots, indicates the presence of three retention behaviors happening in UHPSFC when switching from high to low temperatures, depending on the co-solvent percentage needed to elute one analyte. Finally, an assessment of the stationary phase stability at high temperatures was performed: the retention times variabilities recorded were minimal (RSD < 2.5%), as well as the peak widths and inlet column pressures were somewhat constant throughout the analyses. In the second part of this study, a focus on potential applications benefiting from such unconventional temperatures has been made. A series of challenging analytes have experienced better chromatographic resolution at either high or low temperatures, providing therefore a potentially interesting tool to analysts during the chromatographic method development process. In conclusion, the UV sensitivity at different temperatures was also taken into consideration, with no significant impact on the quality of the UV signal under any condition.
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http://dx.doi.org/10.1016/j.aca.2020.07.076DOI Listing
October 2020

Development of an innovative salt-mediated pH gradient cation exchange chromatography method for the characterization of therapeutic antibodies.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Dec 7;1160:122379. Epub 2020 Sep 7.

Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address:

The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.
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http://dx.doi.org/10.1016/j.jchromb.2020.122379DOI Listing
December 2020

Therapeutic Fc-fusion proteins: Current analytical strategies.

J Sep Sci 2021 Jan 29;44(1):35-62. Epub 2020 Sep 29.

School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of isoelectric points and complex glycosylation profiles including multiple N- and O-linked glycosylation sites. In this review, we highlight the wide range of analytical strategies used to fully characterize Fc-fusion proteins. We also present case studies on the structural assessment of all commercially available Fc-fusion proteins, based on the features and critical quality attributes of their ligand-binding domains.
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http://dx.doi.org/10.1002/jssc.202000765DOI Listing
January 2021

Targeted Bottom-up Characterization of Recombinant Monoclonal Antibodies by Multidimensional LC/MS.

Anal Chem 2020 10 18;92(19):13420-13426. Epub 2020 Sep 18.

Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States.

On-line bottom-up approaches have recently emerged as promising alternatives to standard off-line processes for characterizing post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs). The benefits of on-line processing include reductions in required sample amount and sample handling, as well as reducing the overall turnaround time. However, shortening digestion time for the on-line approach of an intact mAb can cause incomplete peptide cleavages, leading to low sequence coverage and poor repeatability of analyses. For the first time, we describe a novel, automated targeted bottom-up strategy consisting of reducing the complexity of intact mAb by digesting the product into small ∼25 kDa fragments, followed by an on-line peptide mapping analysis of each fragment. For this purpose, a four-dimensional-liquid chromatography/mass spectrometry (4D-LC/MS) method was developed using an immobilized -high-performance liquid chromatography (HPLC) column as a first dimension (D) for on-line digestion, followed by a (D) on-column reversed-phase liquid chromatography (RPLC) for reduction and fragments separation. Then, only one fragment was selected for digestion using a (D) immobilized trypsin cartridge and, finally, the obtained peptides were analyzed by (D) RPLC-MS. This strategy considerably improved the on-line digestion efficiency with higher sequence coverages (LC and HC >97%), thus allowing various PTMs including oxidation, deamidation, and isomerization located in the complementarity-determining regions (CDRs), as well as -glycans present on the Fc/2 fragment, to be monitored with similar sensitivity to those obtained with standard off-line approaches. Additional investigations at a middle-up level were also performed via a three-dimensional-LC/MS (3D-LC/MS) approach within the same system, demonstrating the feasibility to achieve a multilevel comprehensive characterization of mAbs.
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http://dx.doi.org/10.1021/acs.analchem.0c02780DOI Listing
October 2020

Impact of the column on effluent pH in cation exchange pH gradient chromatography, a practical study.

J Chromatogr A 2020 Aug 17;1626:461350. Epub 2020 Jun 17.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland. Electronic address:

In ionexchange chromatography, the pH gradient mode becomes more and more popular today for the analysis of therapeutic proteins as this mode can provide higher or alternative selectivity to the commonly used salt gradient mode. Ideally, a linear pH response is expected when performing linear gradients. However up to now, only a very few buffer systems have been developed and are commercially available which can perform nearly linear pH responses when flowing through a given column. It is also known that a selected buffer system (mobile phase) can work well on one column but can fail on other column. The goal of this study was to practically evaluate the effects that ionexchange columns (weak and strong exchangers) might have on effluent pH, when performing linear pH gradient separations of therapeutic monoclonal antibodies. To attain this objective, the pH was monitored on-line at the column outlet using a specific setup. To make comprehensive observations of the phenomenon, four different mobile phase conditions and five cation exchange columns (weak and strong exchangers) were employed. The obtained pH responses were systematically compared to responses measured in the absence of the columns. From this work, it has become clear that both the column and mobile phase can have significant effects on pH gradient chromatography and that their combination must be considered when developing a new method. Phase systems (column + mobile phase) providing linear pH responses are indeed the most suitable for separating mAbs with different isoelectric points and, with them, it is possible to elute mAbs across wide retention time ranges and with high selectivity.
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http://dx.doi.org/10.1016/j.chroma.2020.461350DOI Listing
August 2020

Fast and Automated Characterization of Monoclonal Antibody Minor Variants from Cell Cultures by Combined Protein-A and Multidimensional LC/MS Methodologies.

Anal Chem 2020 06 3;92(12):8506-8513. Epub 2020 Jun 3.

Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States.

Monitoring of post-translational modifications (PTMs) in therapeutic monoclonal antibodies (mAbs) is essential during their production in both upstream and downstream processes. However, characterization of PTMs using a conventional peptide mapping procedure requires time-consuming and labor-intensive offline sample preparation steps. This work describes for the first time, the implementation of a Protein-A affinity chromatography column as the first dimension (D) in a multidimensional LC (3D and 4D) setup for the automated characterization of mAb variants from harvest cell culture fluid (HCCF) materials at different purification/production steps. A 4D-LC/MS method (Protein-A-Reduction-RPLC-Digestion-RPLC/MS) was developed to determine PTM levels including oxidation, deamidation, and succinimide formation by online peptide mapping analysis. To obtain an accurate and comprehensive profiling of mAb glycosylation patterns at the reduced level, a 3D-LC/MS method (Protein-A-Reduction-RPLC-HILIC/MS) was also developed on the same chromatographic system. Overall, the full workflow (data acquisition and analysis) for both 3D and 4D-LC/MS setups can be completed within less than 1-2 days, compared to weeks with the conventional manual approach. This proof of concept study demonstrates that mD-LC/MS has the potential to be used as a powerful tool to perform a fast and reliable monitoring of PTMs during the manufacturing process for both bioreactor control or as a monitoring assay.
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http://dx.doi.org/10.1021/acs.analchem.0c01250DOI Listing
June 2020

Glycan-Mediated Technology for Obtaining Homogeneous Site-Specific Conjugated Antibody-Drug Conjugates: Synthesis and Analytical Characterization by Using Complementary Middle-up LC/HRMS Analysis.

Anal Chem 2020 06 29;92(12):8170-8177. Epub 2020 May 29.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

Conventional antibody-drug conjugate (ADC) manufacturing methods are based on the nonselective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug-antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice. Here, we evaluated the conjugation process by comparing trastuzumab and trastuzumab conjugated with DM1, following the GlyCLICK procedure. Complementary reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS) were used to analyze the protein subunits (ca. 25-100 kDa) obtained after different levels of enzymatic digestion and chemical reduction. Our results demonstrated that the hydrophobic character of the drug molecule allows to rapidly confirm the Fc-drug conjugation at the chromatographic level. Furthermore, the hyphenation to MS detection provided accurate mass information on the ADC subunits and facilitated the DAR determination of 2.0. Therefore, this work illustrates how middle-up analysis using LC/HRMS can provide accurate and complementary information on the critical quality attributes of these novel site-specific ADC products.
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http://dx.doi.org/10.1021/acs.analchem.0c00282DOI Listing
June 2020

Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices.

J Chromatogr A 2020 Jun 7;1620:461021. Epub 2020 Mar 7.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks. In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%).
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http://dx.doi.org/10.1016/j.chroma.2020.461021DOI Listing
June 2020
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