Publications by authors named "Cinzia Stella"

24 Publications

  • Page 1 of 1

Discovery of a dual pathway aggregation mechanism for a therapeutic constrained peptide.

J Pharm Sci 2021 06 27;110(6):2362-2371. Epub 2021 Feb 27.

Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.
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http://dx.doi.org/10.1016/j.xphs.2020.12.041DOI Listing
June 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

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

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

Immunoaffinity LC-MS/MS is more suitable than ELISA to quantify a PEGylated molecule in cynomolgus monkey serum.

Bioanalysis 2020 Aug 31;12(15):1061-1069. Epub 2020 Jul 31.

Department of Bioanalytical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Polyethylene glycolylation (PEGylation) technology is a long-acting delivery platform used to increase the half-life of protein therapeutics. Quantitation of PEGylated anti-Factor D Fab (PEG-aFD) poses bioanalytical challenges. An ELISA was developed to determine total Fab concentration in cynomolgus monkey serum following intravitreal administration of PEG-aFD. However, assay characterization showed a low recovery of about 25% for free unconjugated Fab whereas recovery for PEG-conjugated Fab was within 80-120%. To overcome this challenge, an immunoaffinity liquid chromatography tandem mass spectrometry (IA LC-MS/MS) assay was developed, achieving recovery within 80-120% for both free and conjugated Fab. Immunoaffinity LC-MS/MS is more suitable than ELISA to accurately quantify the total protein concentration of PEG-aFD in cynomolgus monkey serum.
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http://dx.doi.org/10.4155/bio-2020-0097DOI 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

Development of a 3D-LC/MS Workflow for Fast, Automated, and Effective Characterization of Glycosylation Patterns of Biotherapeutic Products.

Anal Chem 2020 03 4;92(6):4357-4363. Epub 2020 Mar 4.

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

Glycosylation is a common post-translational modification of therapeutic monoclonal antibodies produced in mammalian cells and is considered an important critical quality attribute (CQA), as it is known to impact efficacy, stability, half-life, and immunogenicity. For these reasons, glycosylation requires characterization and close monitoring during the manufacturing process. Due to the complexity of the glycosylation patterns, sophisticated analytical tools with high resolving power are required for the characterization of the glycoforms. This study describes, for the first time, the development and use of an online three-dimensional high-performance liquid chromatography/mass spectrometry (3D-HPLC/MS) approach for the monitoring of glycosylation patterns at the middle-up level. An immobilized IdeS-enzyme column was used in the first dimension for the digestion of mAbs in 10 min. Then, following an online reversed phase liquid chromatography (RPLC) column reduction, the ≈25 kDa proteolytic fragments were analyzed using hydrophilic interaction chromatography (HILIC) coupled to MS. This novel analytical workflow demonstrated the ability to accurately profile glycosylated variants within a total run time of 95 min. To compare the performance of this analytical strategy with a conventional offline procedure (IdeS digestion x reduction-HILIC/MS), a proof of concept study using two mAbs is described here.
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http://dx.doi.org/10.1021/acs.analchem.9b05193DOI Listing
March 2020

Automated middle-up approach for the characterization of biotherapeutic products by combining on-line hinge-specific digestion with RPLC-HRMS analysis.

J Pharm Biomed Anal 2020 Apr 25;182:113130. Epub 2020 Jan 25.

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

The development of biotherapeutic proteins requires the use of efficient analytical methods to support their manufacturing process and quality control (QC). Analytical approaches at intact and middle-up levels emerged as promising alternatives to bottom-up strategies for protein characterization as they require less sample amount and simplified sample handling, thus reducing the overall turn-around time. This study describes, for the first time, the development of an automated liquid chromatography-mass spectrometry (LC-MS) workflow comprised of an immobilized IdeS-HPLC column for on-line sample digestion, followed by a reversed-phase liquid chromatography (RPLC) for protein subunit separation, and a high-resolution MS for molecular weight analysis. A proof of concept study was described here for the characterization of a therapeutic mAb and a bsAb. For the mAb, this automated workflow enabled rapid characterization of post-translational modifications (PTMs) such as N-glycosylation, glycation and N-terminal lysine. For the bsAb, the same workflow was successfully employed to identify product impurities due to chain pairing. The sample analysis using this workflow can be accomplished within less than one day. This workflow demonstrated its potential as a multi-attribute method for characterization of therapeutic proteins.
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http://dx.doi.org/10.1016/j.jpba.2020.113130DOI Listing
April 2020

From proof of concept to the routine use of an automated and robust multi-dimensional liquid chromatography mass spectrometry workflow applied for the charge variant characterization of therapeutic antibodies.

J Chromatogr A 2020 Mar 27;1615:460740. Epub 2019 Nov 27.

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

The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from D CEX analysis were consecutively reduced on a D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1-2 min using a D immobilized trypsin cartridge, and finally the obtained peptides were separated on another D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. D RPLC columns and D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of D and D RPLC column temperature, and D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure.
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http://dx.doi.org/10.1016/j.chroma.2019.460740DOI Listing
March 2020

Immunoaffinity LC-MS/MS to quantify a PEGylated anti-Factor D Fab biotherapeutic in cynomolgus monkey serum.

Bioanalysis 2019 Dec 8;11(23):2161-2173. Epub 2019 Nov 8.

Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.
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http://dx.doi.org/10.4155/bio-2019-0082DOI Listing
December 2019

Streamlined Characterization of an Antibody-Drug Conjugate by Two-Dimensional and Four-Dimensional Liquid Chromatography/Mass Spectrometry.

Anal Chem 2019 12 30;91(23):14896-14903. Epub 2019 Oct 30.

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

This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody-drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5-3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches.
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http://dx.doi.org/10.1021/acs.analchem.9b02454DOI Listing
December 2019

Statistical characterization of therapeutic protein modifications.

Sci Rep 2017 08 11;7(1):7896. Epub 2017 Aug 11.

Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.

Peptide mapping with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an important analytical method for characterization of post-translational and chemical modifications in therapeutic proteins. Despite its importance, there is currently no consensus on the statistical analysis of the resulting data. In this manuscript, we distinguish three statistical goals for therapeutic protein characterization: (1) estimation of site occupancy of modifications in one condition, (2) detection of differential site occupancy between conditions, and (3) estimation of combined site occupancy across multiple modification sites. We propose an approach, which addresses these goals in terms of summarizing the quantitative information from the mass spectra, statistical modeling, and model-based analysis of LC-MS/MS data. We illustrate the approach using an LC-MS/MS experiment from an antibody-drug conjugate and its monoclonal antibody intermediate. The performance was compared to a 'naïve' data analysis approach, by using computer simulation, evaluation of differential site occupancy in positive and negative controls, and comparisons of estimated site occupancy with orthogonal experimental measurements of N-linked glycoforms and total oxidation. The results demonstrated the importance of replicated studies of protein characterization, and of appropriate statistical modeling, for reproducible, accurate and efficient site occupancy estimation and differential analysis.
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http://dx.doi.org/10.1038/s41598-017-08333-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554216PMC
August 2017

Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Oct 14;1032:39-50. Epub 2016 Jul 14.

Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jchromb.2016.07.023DOI Listing
October 2016

Investigation of low recovery in the free drug assay for antibody drug conjugates by size exclusion-reversed phase two dimensional-liquid chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Oct 10;1032:112-118. Epub 2016 May 10.

Small Molecule Pharmaceutical Sciences, Genentech, South San Francisco, CA 94080, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jchromb.2016.05.011DOI Listing
October 2016

Characterization of oxidative carbonylation on recombinant monoclonal antibodies.

Anal Chem 2014 May 30;86(10):4799-806. Epub 2014 Apr 30.

Protein Analytical Chemistry, ‡X-ray Laboratory, Genentech Inc. , South San Francisco, California 94080, United States.

In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry.
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http://dx.doi.org/10.1021/ac4039866DOI Listing
May 2014

Identification of human urinary biomarkers of cruciferous vegetable consumption by metabonomic profiling.

J Proteome Res 2011 Oct 31;10(10):4513-21. Epub 2011 Aug 31.

Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.

Consumption of cruciferous vegetables (CVs) is inversely correlated to many human diseases including cancer (breast, lung, and bladder), diabetes, and cardiovascular and neurological disease. Presently, there are no readily measurable biomarkers of CV consumption and intake of CVs has relied on dietary recall. Here, biomarkers of CV intake were identified in the urine of 20 healthy Caucasian adult males using (1)H NMR spectroscopy with multivariate statistical modeling. The study was separated into three phases of 14 days: a run-in period with restricted CV consumption (phase I); a high CV phase where participants consumed 250 g/day of both broccoli and Brussels sprouts (phase II); a wash-out phase with a return to restricted CV consumption (phase III). Each study participant provided a complete cumulative urine collection over 48 h at the end of each phase; a spot urine (U0), 0-10 h (U0-10), 10-24 h (U10-24), and 24-48 h (U24-48) urine samples. Urine samples obtained after consumption of CVs were differentiated from low CV diet samples by four singlet (1)H NMR spectroscopic peaks, one of which was identified as S-methyl-l-cysteine sulfoxide (SMCSO) and the three other peaks were tentatively identified as other metabolites structurally related to SMCSO. These stable urinary biomarkers of CV consumption will facilitate future assessment of CVs in nutritional population screening and dietary intervention studies and may correlate to population health outcomes.
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http://dx.doi.org/10.1021/pr200326kDOI Listing
October 2011

Association of ranibizumab (Lucentis®) or bevacizumab (Avastin®) with dexamethasone and triamcinolone acetonide: an in vitro stability assessment.

Eur J Pharm Biopharm 2011 Jun 21;78(2):271-7. Epub 2010 Dec 21.

Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.

The in vitro stability of monoclonal antibodies used for age-related macular degeneration, ranibizumab and bevacizumab, was investigated. The aggregation profile of the antibodies was compared, alone and after association with dexamethasone sodium phosphate or triamcinolone acetonide. Commercial formulations of ranibizumab and bevacizumab were dialysed into three different buffers. After dialysis, samples were stored at 4°C, 25°C and 40°C during 35 days, alone and in combination with dexamethasone sodium phosphate, triamcinolone acetonide phosphate solution or triamcinolone acetonide suspension. Combined formulations based on both commercial formulations were investigated as well. The aggregation state of the antibodies was measured by multi-angle light scattering (MALS) after separation by asymmetrical flow field-flow fractionation (AFFF) or size-exclusion chromatography (SEC). Ranibizumab results to be more stable than bevacizumab, alone and in combination with dexamethasone sodium phosphate or triamcinolone acetonide. Elevation in concentration, pH and temperature causes a decrease in stability of both antibodies. The association of triamcinolone acetonide phosphate solution with either ranibizumab or bevacizumab is observed to be the least stable combination of all samples tested. Dexamethasone sodium phosphate was shown to have a stabilizing effect on bevacizumab, although this is not the case for its combination with the commercial formulation Avastin®. The results demonstrate that the in vitro association of either ranibizumab or bevacizumab with dexamethasone sodium phosphate or triamcinolone acetonide suspension does not decrease the stability of these antibodies. Although ranibizumab is more stable than bevacizumab in vitro, further research has to point out how this affects their mechanism of action in vivo.
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http://dx.doi.org/10.1016/j.ejpb.2010.12.018DOI Listing
June 2011

Liquid chromatography-mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies.

Biomed Chromatogr 2010 Jul;24(7):737-43

Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, South Kensington, London, UK.

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC-MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high-fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid-liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial-least-squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid-liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class-based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects.
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http://dx.doi.org/10.1002/bmc.1357DOI Listing
July 2010

Pharmacokinetics and posterior segment biodistribution of ESBA105, an anti-TNF-alpha single-chain antibody, upon topical administration to the rabbit eye.

Invest Ophthalmol Vis Sci 2009 Feb 29;50(2):771-8. Epub 2008 Aug 29.

ESBATech AG, Schlieren, Switzerland.

Purpose: The purpose of this study was to characterize local distribution and systemic absorption of the tumor necrosis factor (TNF)-alpha inhibitory single-chain antibody fragment (scFv) ESBA105 following topical administration to the eye in vivo.

Methods: Rabbits received ESBA105 as topical eye drops in two dosing regimens. First, pharmacokinetics after the topical route of administration was compared to the intravenous (i.v.) route by means of applying the identical cumulative daily dose of ESBA105. In a second study rabbits received five eye drops daily for six consecutive days in a lower frequency topical dosing regimen. Kinetics and biodistribution of ESBA105 in ocular tissues and fluids as well as in sera were determined in all animals.

Results: After topical administration to the eye, ESBA105 quickly reaches therapeutic concentrations in all ocular compartments. Systemic exposure after topical administration is 25,000-fold lower than exposure after i.v. injection of the identical cumulative daily dose. ESBA105 levels in vitreous humor and neuroretina are significantly higher on topical administration than after i.v. injection. Absolute and relative intraocular biodistribution of ESBA105 is different with topical and systemic delivery routes. Compared to its terminal half-life in circulation (7 hours), the vitreal half-life of ESBA105 is significantly enhanced (16-24 hours).

Conclusions: On topical administration, ESBA105 is efficiently absorbed and distributed to all compartments of the eye, whereby systemic drug exposure is very low. Based on its unique intraocular biodistribution and pharmacokinetics and the absolute intraocular levels reached, topical ESBA105 appears highly attractive for treatment of various ophthalmological disorders.
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http://dx.doi.org/10.1167/iovs.08-2370DOI Listing
February 2009

Susceptibility of human metabolic phenotypes to dietary modulation.

J Proteome Res 2006 Oct;5(10):2780-8

Biomolecular Medicine, Division of Surgery, Oncology, Reproductive Biology and Anaesthetics (SORA), Faculty of Medicine, Imperial College London, and MRC Dunn Human Nutrition Unit, Addenbrookes Hospital, Cambridge, United Kingdom.

Dietary composition has been shown to influence metabolism and to impact on the prevalence and risk for certain diseases, but hitherto, there have been no systematic studies on the effects of dietary modulation of human metabolic phenotype (metabotype). Here, we have applied 1H NMR spectroscopy in combination with multivariate statistical analysis to characterize the effects of three diets: "vegetarian", "low meat", and "high meat" on the metabotype signature of human participants. Twelve healthy male participants (age range of 25-74 years) consumed each of these diets, in a randomized order, for continuous 15-day-periods with an intervening washout period between each diet of 7 days duration. Each participant provided three consecutive 24-hour urine collections on days 13, 14, and 15 of each dietary period, and 1H NMR spectra were acquired on all samples. Pattern recognition analysis allowed differentiation of the characteristic metabolic signatures of the diets with creatine, carnitine, acetylcarnitine, and trimethylamine-N-oxide (TMAO) being elevated in the high-meat consumption period. Application of orthogonal projection to latent structure discriminant analysis (O-PLS-DA) allowed the low-meat diet and vegetarian diet signatures to be characterized, and p-hydroxyphenylacetate (a microbial mammalian cometabolite) was higher in the vegetarian than meat diet samples, signaling an alteration of the bacterial composition or metabolism in response to diet. This work shows the potential for the routine use of metabonomics in nutritional and epidemiological studies, in characterizing and predicting the metabolic effects and the influence of diet on human metabotypes.
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http://dx.doi.org/10.1021/pr060265yDOI Listing
October 2006

Characterization and comparison of the chromatographic performance of different types of reversed-phase stationary phases.

J Pharm Biomed Anal 2007 Jan 24;43(1):89-98. Epub 2006 Jul 24.

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, University of Geneva, Switzerland.

The chromatographic performance of several base-deactivated stationary phases was evaluated with a specific chromatographic test. Seven basic test compounds, possessing different physico-chemical properties were injected on different supports with two mobile phases: one at pH 7.0 (acetonitrile-phosphate buffer, 40:60, v/v), and the other at pH 3.0 (acetonitrile-phosphate buffer, 15:85, v/v). Chromatographic parameters obtained under these conditions were treated by principal component analysis (PCA) to separate base deactivated supports according to their silanol activity (pH 7.0 mobile phase) and hydrophobic properties (pH 3.0 mobile phase). The information given by the specific test column evaluation was improved with complementary chemometric tools such as hierarchical cluster analysis. The same base deactivated supports were also tested following a general test procedure issued from the literature and obtained fundamental properties (in particular silanol activity and hydrophobicity) were compared with column evaluation obtained with the specific test: results were in good agreement, although the use of the specific test offered a better differentiation between numerous base-deactivated supports.
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http://dx.doi.org/10.1016/j.jpba.2006.06.018DOI Listing
January 2007

Novel RPLC stationary phases for lipophilicity measurement: solvatochromic analysis of retention mechanisms for neutral and basic compounds.

J Sep Sci 2005 Nov;28(17):2350-62

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, EPGL, University of Geneva, Switzerland.

An RPLC was developed to rapidly determine lipophilicity of neutral and basic compounds using three base deactivated RPLC stationary phases particularly designed for the analysis of basic compounds, namely, Supelcosil ABZ(+)Plus, Discovery RP Amide C16, and Zorbax Extend C18. The work consisted of three sets of experiments. In the first log kw values of neutral compounds were extrapolated using hydroorganic mobile phases at different compositions. Good correlation between log kw and log Poct indicated that the method was appropriate for these supports, without adding a silanol masking agent. In the second set of experiments, isocratic log k values of neutral and basic compounds were measured with three different mobile phases. The best estimation of lipophilicity was obtained for neutral and basic compounds when the secondary interactions were strongly reduced (i. e., when basic compounds were under their neutral form). In the third set of experiments, isocratic retention factors of basic compounds (in their neutral form) were measured with a high-pH mobile phase, on a chemically stable support (Zorbax Extend C18). Under these chromatographic conditions, correlation between the isocratic retention factors and log Poct (log D10.5) for basic compounds was similar to that for neutral compounds.
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http://dx.doi.org/10.1002/jssc.200500104DOI Listing
November 2005

A study of interlaboratory influence on column evaluation.

J Pharm Biomed Anal 2005 Sep;39(1-2):104-10

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, EPGL, University of Geneva, Switzerland.

A liquid chromatography method for the characterization of base deactivated columns was investigated in a collaborative study involving six laboratories. This work was carried out on two chromatographic supports (Xterra RP 18 and Symmetry Shield). Different cooling systems, namely water bath and air oven, were tested and it was shown that column thermoregulation did not significantly influence chromatographic data. In order to control the mobile phase composition, the latter was prepared by weight rather than volume. Thanks to the injection of a set of selected neutral compounds, extra-column effects were evaluated in each of the participating laboratories. The results showed that chromatographic supports tested in different laboratories and following the same test protocol could be effectively compared.
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http://dx.doi.org/10.1016/j.jpba.2005.03.007DOI Listing
September 2005

Analysis of basic compounds at high pH values by reversed-phase liquid chromatography.

J Sep Sci 2004 Mar;27(4):284-92

Laboratory of Pharmaceutical Analytical Chemistry - School of Pharmacy - University of Geneva, 1211 Geneva 4, Switzerland.

Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C18, was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.
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http://dx.doi.org/10.1002/jssc.200301671DOI Listing
March 2004