20 results on '"Barry Lorbetskie"'
Search Results
2. Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems
- Author
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Daryl G.S. Smith, Grant E. Frahm, Anita Kane, Barry Lorbetskie, Michel Girard, Michael J.W. Johnston, and Terry D. Cyr
- Subjects
Albumin ,Recombinant proteins ,Glycation ,Mass spectrometry ,Oryza sativa ,Computer applications to medicine. Medical informatics ,R858-859.7 ,Science (General) ,Q1-390 - Abstract
Human serum albumin (HSA) is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3) (2012) 209–290). Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA) (Chen et al., Biochim. Biophys. Acta (BBA)—Gen. Subj. 1830(12) (2013) 5515–5525; Kobayashi, Biologicals 34(1) (2006) 55–59). Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15) (2012) 4661–4670), both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9) (2014) e109893). We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.
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- 2015
- Full Text
- View/download PDF
3. Nanoparticle size and production efficiency are affected by the presence of fatty acids during albumin nanoparticle fabrication.
- Author
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Christian C Luebbert, Tessa M Clarke, Roberta Pointet, Grant E Frahm, Sharon Tam, Barry Lorbetskie, Simon Sauvé, and Michael J W Johnston
- Subjects
Medicine ,Science - Abstract
We have previously identified extensive glycation, bound fatty acids and increased quantities of protein aggregates in commercially available recombinant HSA (rHSA) expressed in Oryza sativa (Asian rice) (OsrHSA) when compared to rHSA from other expression systems. We propose these differences may alter some attributes of nanoparticles fabricated with OsrHSA, as studies have associated greater quantities of aggregates with increased nanoparticle diameters. To determine if this is the case, nanoparticles were fabricated with OsrHSA from various suppliers using ethanol desolvation and subsequent glutaraldehyde cross-linking. All nanoparticles fabricated with OsrHSA showed larger diameters of approximately 20 to 90nm than particles fabricated with either defatted bovine serum albumin (DF-BSA) (100.9 ± 2.8nm) or human plasma albumin (pHSA) (112.0 ± 4.0nm). It was hypothesized that the larger nanoparticle diameters were due to the presence of bound fatty acids and this was confirmed through defatting OsrHSA prior to particle fabrication which yielded particles with diameters similar to those fabricated with pHSA. For additional conformation, DF-BSA was incubated with dodecanoic acid prior to desolvation yielding particles with significantly larger diameters. Further studies showed the increased nanoparticle diameters were due to the bound fatty acids modulating electrostatic interactions between albumin nanoparticles during the desolvation and not changes in protein structure, stability or generation of additional albumin oligomers. Finally the presence of dodecanoic acid was shown to improve doxorubicin loading efficiency onto preformed albumin nanoparticles.
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- 2017
- Full Text
- View/download PDF
4. Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.
- Author
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Grant E Frahm, Daryl G S Smith, Anita Kane, Barry Lorbetskie, Terry D Cyr, Michel Girard, and Michael J W Johnston
- Subjects
Medicine ,Science - Abstract
The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.
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- 2014
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5. Regulatory Verification by Health Canada of Content in Recombinant Human Insulin, Human Insulin Analog, and Porcine Insulin Drug Products in the Canadian Market Using Validated Pharmacopoeial Methods Over Nonvalidated Approaches
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Barry Lorbetskie, Stewart Bigelow, Lisa Walrond, Agnes V. Klein, Shih-Miin Loo, Nancy Green, Michael Rosu-Myles, Xu Zhang, Huixin Lu, Michel Girard, and Simon Sauvé
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Endocrinology, Diabetes and Metabolism ,Biomedical Engineering ,Internal Medicine ,Bioengineering - Abstract
Background: For diabetes mellitus treatment plans, the consistency and quality of insulin drug products are crucial for patient well-being. Because biologic drugs, such as insulin, are complex heterogeneous products, the methods for drug product evaluation should be carefully validated for use. As such, these criteria are rigorously evaluated and monitored by national authorities. Consequently, reports that describe significantly lower insulin content than their label claims are a concern. This issue was raised by a past publication analyzing insulin drug products available in Canada, and, as a result, consumers and major patient organizations have requested clarification. Methods: To address these concerns, this study independently analyzed insulin drug products purchased from local Canadian pharmacies—including human insulin, insulin analogs, and porcine insulin—by compendial and noncompendial reversed-phase high-performance liquid chromatography (RP-HPLC) methods. Results: We demonstrated the importance of using methods fit for purpose when assessing insulin quality. In a preliminary screen, the expected insulin peak was seen in all products except two insulin analogs—insulin detemir and insulin degludec. Further investigation showed that this was not caused by low insulin content but insufficient solvent conditions, which demonstrated the necessity for methods to be adequately validated for product-specific use. When drug products were appropriately assessed for content using the validated type-specific compendial RP-HPLC methods for insulin quantitation, values agreed with the label claim content. Conclusions: Because insulin drug products are used daily by over a million Canadians, it is important that researchers and journals present data using methods fit for purpose and that readers evaluate such reports critically.
- Published
- 2023
6. Selective reversed-phase high-performance liquid chromatography method for the determination of intact SARS-CoV-2 spike protein
- Author
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Barry Lorbetskie, Taryn White, Marybeth Creskey, Xu Zhang, Michel Girard, Roger Y. Tam, Simon Sauvé, and Huixin Lu
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COVID-19 Vaccines ,SARS-CoV-2 ,Organic Chemistry ,Spike Glycoprotein, Coronavirus ,COVID-19 ,Humans ,General Medicine ,Biochemistry ,Pandemics ,Chromatography, High Pressure Liquid ,Analytical Chemistry - Abstract
Protein-based vaccines are playing an increasingly important role in the COVID-19 pandemic. As late-stage clinical data are finalized and released, the number of protein-based vaccines expected to enter the market will increase significantly. Most protein-based COVID-19 vaccines are based on the SARS-CoV-2 spike protein (S-protein), which plays a major role in viral attachment to human cells and infection. As a result, in order to develop and manufacture quality vaccines consistently, it is imperative to have access to selective and efficient methods for the bioanalytical assessment of S-protein. In this study, samples of recombinant S-protein (hexS-protein) and commercial S-protein were used to develop a selective reversed-phase HPLC (RP-HPLC) method that enabled elution of the intact S-protein monomer as a single peak on a wide pore, C8-bonded chromatographic column. The S-protein subunits, S1 and S2 subunits, were clearly separated from intact S-protein and identified. The results of this study set the foundation for reversed-phase HPLC method development and analysis for selective and efficient separation of S-protein monomer from its subunits.
- Published
- 2021
7. Selective Capture and Determination of Receptor-Binding Hemagglutinin in Influenza Vaccine Preparations Using a Coupled Receptor-Binding/RP-HPLC Assay
- Author
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Aaron Farnsworth, Michel Girard, Laura Durno, Simon Sauvé, Michel Gilbert, Changgui Li, Michèle Lemieux, Xuguang Li, Junzhi Wang, Barry Lorbetskie, and Anna-Maria Cunningham
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Models, Molecular ,assays ,Influenza vaccine ,receptors ,Hemagglutinin (influenza) ,Hemagglutinin Glycoproteins, Influenza Virus ,Sialic acid binding ,medicine.disease_cause ,materials ,Analytical Chemistry ,Birds ,Influenza A Virus, H1N1 Subtype ,Influenza, Human ,Influenza A virus ,medicine ,Animals ,Humans ,Potency ,viruses ,Vaccine Potency ,Chromatography, High Pressure Liquid ,Radial immunodiffusion ,Chromatography, Reverse-Phase ,biology ,Chemistry ,vaccination ,N-Acetylneuraminic Acid ,In vitro ,Vaccines, Inactivated ,Biochemistry ,Influenza Vaccines ,Influenza in Birds ,biology.protein - Abstract
Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.
- Published
- 2019
8. Fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method
- Author
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Simon Sauvé, Changgui Li, Nathalie Fortin, Xuguang Li, Laura Durno, Junzhi Wang, Barry Lorbetskie, and Michel Girard
- Subjects
0301 basic medicine ,Trivalent influenza vaccine ,Chemistry, Pharmaceutical ,Hemagglutinin (influenza) ,medicine.disease_cause ,Biochemistry ,High-performance liquid chromatography ,Virus ,Analytical Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,Influenza A virus ,medicine ,030212 general & internal medicine ,Chromatography, Reverse-Phase ,Chromatography ,biology ,Chemistry ,Strain (biology) ,Organic Chemistry ,General Medicine ,Vaccination ,Influenza B virus ,Hemagglutinins ,030104 developmental biology ,Influenza Vaccines ,biology.protein - Abstract
Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.
- Published
- 2017
9. The differential effect of sub-micron level HA aggregates on influenza potency assays
- Author
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M.J.W. Johnston, Christian C. Luebbert, Simon Sauvé, Michèle Lemieux, Changgui Li, Terry D. Cyr, Aaron Farnsworth, Xuguang Li, Barry Lorbetskie, and Lisa Walrond
- Subjects
Male ,Immunogen ,Influenza vaccine ,030231 tropical medicine ,Blotting, Western ,Hemagglutinin (influenza) ,Enzyme-Linked Immunosorbent Assay ,Biology ,Epitope ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Antigen ,Influenza, Human ,Potency ,Animals ,Humans ,030212 general & internal medicine ,Vaccine Potency ,General Veterinary ,General Immunology and Microbiology ,Public Health, Environmental and Occupational Health ,Virology ,Mice, Inbred C57BL ,Infectious Diseases ,Hemagglutinins ,Influenza Vaccines ,biology.protein ,Molecular Medicine ,Electrophoresis, Polyacrylamide Gel ,Antibody - Abstract
Influenza vaccines remain the most effective public health measure for the prevention of influenza-related illnesses. The primary immunogen in inactivated influenza vaccines is hemagglutinin (HA), the receptor binding protein of influenza. The concentration of HA during vaccine production and testing is standardized according to the level of antigen as measured by Single Radial Immunodiffusion Assay (SRID). This allows vaccine potency to be controlled such that individuals receive a dose known to provoke a clinically protective immune response. As compared to alternatives, SRID has the advantage of quantifying immunologically relevant forms of HA, but it depends on timely generation of novel reagents for each new vaccine strain. In recent years, a number of alternative assays have been suggested based on either epitope recognition, receptor binding or protection from proteolysis but it is unclear how they relate to vaccine potency in clinical trials. In this report we describe the development of a lectin-based, ELISA-type assay for HA potency and find it provides similar potency estimates to SRID except in the case of a vaccine with aggregated HA and other viral proteins. In that case, SRID predicted the immunologically active HA present and ELISA techniques did not. This difference was due to tested antibodies failing to pull down or bind to the HA present unless particle aggregates were first dissociated. Furthermore, detergent treatment alone was insufficient to complete this dissociation. While others have previously demonstrated that immunocapture-based techniques can misestimate the potency of influenza vaccines depending on the individual antibodies used we demonstrate that in this case the failure was due to an inability of all antibodies to capture HA contained in the aggregated influenza vaccine.
- Published
- 2019
10. Nanoparticle size and production efficiency are affected by the presence of fatty acids during albumin nanoparticle fabrication
- Author
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Simon Sauvé, Christian C. Luebbert, Grant E. Frahm, Michael V. Johnston, Roberta Pointet, Tessa M. Clarke, Barry Lorbetskie, and Sharon Tam
- Subjects
0301 basic medicine ,Circular dichroism ,lcsh:Medicine ,Nanoparticle ,02 engineering and technology ,Protein aggregation ,Biochemistry ,chemistry.chemical_compound ,Macromolecular Structure Analysis ,Nanotechnology ,Electron Microscopy ,Bovine serum albumin ,lcsh:Science ,Microscopy ,Multidisciplinary ,biology ,Chemistry ,Organic Compounds ,Circular Dichroism ,Fatty Acids ,Eukaryota ,Plants ,021001 nanoscience & nanotechnology ,Lipids ,Recombinant Proteins ,Experimental Organism Systems ,Physical Sciences ,Chromatography, Gel ,Engineering and Technology ,Alpha Helix ,Scanning Electron Microscopy ,0210 nano-technology ,Research Article ,Protein Structure ,Serum albumin ,Research and Analysis Methods ,03 medical and health sciences ,Plant and Algal Models ,Albumins ,Humans ,Grasses ,Particle Size ,Molecular Biology ,Serum Albumin ,Chromatography ,Ethanol ,lcsh:R ,Organic Chemistry ,Albumin ,Chemical Compounds ,Organisms ,Biology and Life Sciences ,Proteins ,030104 developmental biology ,Glutaral ,Alcohols ,biology.protein ,Microscopy, Electron, Scanning ,Nanoparticles ,lcsh:Q ,Particle size ,Glutaraldehyde ,Rice - Abstract
We have previously identified extensive glycation, bound fatty acids and increased quantities of protein aggregates in commercially available recombinant HSA (rHSA) expressed in Oryza sativa (Asian rice) (OsrHSA) when compared to rHSA from other expression systems. We propose these differences may alter some attributes of nanoparticles fabricated with OsrHSA, as studies have associated greater quantities of aggregates with increased nanoparticle diameters. To determine if this is the case, nanoparticles were fabricated with OsrHSA from various suppliers using ethanol desolvation and subsequent glutaraldehyde cross-linking. All nanoparticles fabricated with OsrHSA showed larger diameters of approximately 20 to 90nm than particles fabricated with either defatted bovine serum albumin (DF-BSA) (100.9 ± 2.8nm) or human plasma albumin (pHSA) (112.0 ± 4.0nm). It was hypothesized that the larger nanoparticle diameters were due to the presence of bound fatty acids and this was confirmed through defatting OsrHSA prior to particle fabrication which yielded particles with diameters similar to those fabricated with pHSA. For additional conformation, DF-BSA was incubated with dodecanoic acid prior to desolvation yielding particles with significantly larger diameters. Further studies showed the increased nanoparticle diameters were due to the bound fatty acids modulating electrostatic interactions between albumin nanoparticles during the desolvation and not changes in protein structure, stability or generation of additional albumin oligomers. Finally the presence of dodecanoic acid was shown to improve doxorubicin loading efficiency onto preformed albumin nanoparticles.
- Published
- 2017
11. Improved detection of variants in recombinant human interferon alpha-2a products by reverse-phase high-performance liquid chromatography on a core–shell stationary phase
- Author
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Michel Girard, Junzhi Wang, Chunming Rao, Yonghong Li, Lei Tao, and Barry Lorbetskie
- Subjects
Quality Control ,Chemistry, Pharmaceutical ,Clinical Biochemistry ,DNA, Recombinant ,Pharmaceutical Science ,Alpha interferon ,Interferon alpha-2 ,Tandem mass spectrometry ,High-performance liquid chromatography ,Chemistry Techniques, Analytical ,Analytical Chemistry ,law.invention ,chemistry.chemical_compound ,Methionine ,Tandem Mass Spectrometry ,law ,Phase (matter) ,Drug Discovery ,Trifluoroacetic acid ,Humans ,Chromatography, High Pressure Liquid ,Spectroscopy ,Chromatography ,Elution ,Chemistry ,Temperature ,Interferon-alpha ,Reproducibility of Results ,Fluorescence ,Recombinant Proteins ,Protein Structure, Tertiary ,Oxygen ,Recombinant DNA ,Interferons ,Peptides - Abstract
The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core-shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.
- Published
- 2014
12. Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC–MSE
- Author
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Aaron Farnsworth, Terry D. Cyr, Daryl G.S. Smith, Barry Lorbetskie, Junzhi Wang, Changgui Li, Xuguang Li, Marybeth Creskey, Michel Girard, and Caroline Gravel
- Subjects
Influenza vaccine ,Egg protein ,Neuraminidase ,Hemagglutinin (influenza) ,Hemagglutinin Glycoproteins, Influenza Virus ,Mass Spectrometry ,Antigen ,Animals ,Radial immunodiffusion ,General Veterinary ,General Immunology and Microbiology ,biology ,Egg Proteins ,Public Health, Environmental and Occupational Health ,Reproducibility of Results ,Reference Standards ,Virology ,Ovalbumin ,Infectious Diseases ,Influenza Vaccines ,biology.protein ,Molecular Medicine ,Antibody ,Chickens ,Chromatography, Liquid - Abstract
a b s t r a c t Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neu- raminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 g/dose. The SRID assays require the availability of strain- specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neu- raminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS E , a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.
- Published
- 2012
13. Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences
- Author
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Yves Aubin, Xuguang Li, Geneviève Gingras, Anwar M. Hashem, Caroline Gravel, Runtao He, Kangwei Xu, Changgui Li, Junzhi Wang, Michel Girard, Barry Lorbetskie, Garry Van Domselaar, and Bozena Jaentschke
- Subjects
Neuraminidase ,Cross Reactions ,Biology ,Antibodies, Viral ,Sialidase ,Epitope ,Conserved sequence ,Immune system ,Antibody Specificity ,Viral neuraminidase ,Conserved Sequence ,Sequence Homology, Amino Acid ,General Veterinary ,General Immunology and Microbiology ,Public Health, Environmental and Occupational Health ,Antibodies, Monoclonal ,Virology ,Influenza B virus ,Infectious Diseases ,Influenza A virus ,Influenza Vaccines ,biology.protein ,Molecular Medicine ,Viral disease ,Antibody - Abstract
Neuraminidase-induced immune responses are correlated with protection of humans and animals from influenza. However, the amounts of neuraminidase in influenza vaccines are yet to be standardized. Thus, a simple method capable of quantifying neuraminidase would be desirable. Here we identified two universally conserved sequences in all influenza A and B neuraminidases, one representing a novel finding of nearly 100% conservation near the enzymatically active site. Antibodies generated against the two highly conserved sequences bound to all nine subtypes of influenza A neuraminidase and demonstrated remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Importantly, employing these antibodies for the analyses of vaccines from eight manufacturers using the same vaccine seeds revealed marked variations of neuraminidase levels in addition to considerable differences between lots from the same producer. The reasons for the absence or low level of neuraminidase in vaccine preparations are complex and could be multi-factorial. The antibody-based assays reported here could be of practical value for better vaccine quality control.
- Published
- 2010
14. Study of aggregation, denaturation and reduction of interferon alpha-2 products by size-exclusion high-performance liquid chromatography with fluorescence detection and biological assays
- Author
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Abebaw Diress, Xuguang Li, Louise Larocque, Richard Isbrucker, Barry Lorbetskie, Michel Girard, and Matthew Alteen
- Subjects
Protein Denaturation ,Protein Conformation ,Size-exclusion chromatography ,Alpha interferon ,Sensitivity and Specificity ,Biochemistry ,High-performance liquid chromatography ,Analytical Chemistry ,Gel permeation chromatography ,medicine ,Humans ,Denaturation (biochemistry) ,Chromatography, High Pressure Liquid ,Interferon alfa ,Active ingredient ,Chromatography ,Cell Death ,Protein Stability ,Chemistry ,Organic Chemistry ,Temperature ,Reproducibility of Results ,Biological activity ,Hep G2 Cells ,General Medicine ,Recombinant Proteins ,Spectrometry, Fluorescence ,Interferon Type I ,Chromatography, Gel ,Linear Models ,Protein Multimerization ,medicine.drug - Abstract
Interferon alpha-2 (IFN alpha-2) products have been widely used as antivirals for the treatment of serious diseases such as hepatitis B and C. However, reports of adverse reactions following treatment have prompted investigations into the cause of these undesirable events. In this study size-exclusion HPLC (SE-HPLC) methods coupled with intrinsic fluorescence detection were developed for evaluating the stability and degradation profiles of IFN alpha-2 drug substances and drug products. The method allowed baseline resolution of the active ingredient from the excipients present in the finished products that included large amounts of albumin. Limits of detection (S/N>or=3) for IFN alpha-2a and IFN alpha-2b were 32 ng/mL and 28 ng/mL, respectively and good repeatability of chromatographic profiles (%RSD
- Published
- 2010
15. Approach to the profiling and characterization of influenza vaccine constituents by the combined use of size-exclusion chromatography, gel electrophoresis and mass spectrometry
- Author
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Virginia García-Cañas, Barry Lorbetskie, Mary Alice Hefford, Michel Girard, Terry D. Cyr, and Sophie Smith
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Time Factors ,Influenza vaccine ,Size-exclusion chromatography ,Combined use ,Drug Evaluation, Preclinical ,Bioengineering ,Mass spectrometry ,Sensitivity and Specificity ,Applied Microbiology and Biotechnology ,Mass Spectrometry ,Viral Proteins ,Counterfeit Product ,Drug Stability ,Screening method ,Chromatography, High Pressure Liquid ,Pharmacology ,Gel electrophoresis ,Chromatography ,General Immunology and Microbiology ,Chemistry ,Structural integrity ,General Medicine ,Influenza Vaccines ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Chromatography, Gel ,Metabolome ,Electrophoresis, Polyacrylamide Gel ,Protein Processing, Post-Translational ,Biotechnology - Abstract
A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. Size-exclusion HPLC analysis reduced significantly the complexity by providing a constituents profile according to size. Significantly, no sample treatment was required prior to analysis thus eliminating a potential source of artifacts and degradation. Distinct profiles were associated with influenza strains as well as with vaccines from different manufacturers. Samples analyzed over several years allowed evaluation of method performance and provided stability-indicating data relating to the structural integrity of separated components. Collected chromatographic peaks were identified by gel electrophoresis and MALDI/MS of tryptic digests from excised gel bands. The challenge in obtaining high quality analytical data from complex mixtures clearly demonstrated the value of separation steps prior to MS identification. The method presented here is not intended to replace existing methodology; it is intended to provide a product specific profile to be used as a rapid screen for manufacturer, year (for annual influenza vaccines), stability or counterfeit product. It is a new screening method that provides a rapid and robust indication of products which require further investigation as a result of a deviation in their characteristic profile. Until now this tool did not exist.
- Published
- 2010
16. Selective and Quantitative Detection of Influenza Virus Proteins in Commercial Vaccines Using Two-Dimensional High-Performance Liquid Chromatography and Fluorescence Detection
- Author
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Barry Lorbetskie, Michel Girard, Virginia García-Cañas, Terry D. Cyr, and Diane Bertrand
- Subjects
Detection limit ,Chromatography ,biology ,Chemistry ,Influenza vaccine ,Size-exclusion chromatography ,Hemagglutinin (influenza) ,Hemagglutinin Glycoproteins, Influenza Virus ,Reversed-phase chromatography ,Orthomyxoviridae ,Sensitivity and Specificity ,High-performance liquid chromatography ,Mass Spectrometry ,Fluorescence spectroscopy ,Analytical Chemistry ,Influenza Vaccines ,biology.protein ,Quantitative analysis (chemistry) ,Chromatography, High Pressure Liquid - Abstract
In this work, we report on the applicability of two-dimensional high-performance liquid chromatography (2D-HPLC) for the comprehensive characterization of inactivated influenza vaccine proteins. This novel procedure features minimal sample treatment and combines the on-line coupling of size exclusion HPLC to reversed-phase HPLC. A comparative analysis of commercial vaccines from three different manufacturers showed the method to be highly selective by providing characteristic reproducible chromatographic profiles for each vaccine. In addition, the method provided enhanced sensitivity for most constituents as a result of the use of native fluorescence detection in the reversed-phase HPLC step. The limits of detection (at a signal-to-noise ratio of3) for hemagglutinin (HA) antigens were 105 and 172 ng/mL for influenza A/New Caledonia/20/99 and B/Jiangsu/10/2003 strains, respectively. The potential of this 2D-HPLC procedure in terms of quantitative antigen analysis was assessed by determination of the HA content of commercial vaccines. Results provided very good correlation with nominal HA values. The reproducibility (RSD) of the whole procedure was also evaluated and was found to be better than 2 and 3% for calculated antigen concentrations expressed as micrograms of HA per milliliter in commercial vaccines for samples of the same lot (n = 5) or different lots (n = 3), respectively. In addition, it allowed the selective detection of several influenza constituents including nucleoproteins from type A and B viruses and the highly hydrophobic matrix protein 1 from both virus strains.
- Published
- 2007
17. Rapid and selective characterization of influenza virus constituents in monovalent and multivalent preparations using non-porous reversed-phase high performance liquid chromatography columns
- Author
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Virginia García-Cañas, Michel Girard, and Barry Lorbetskie
- Subjects
Protein Denaturation ,Ultraviolet Rays ,Influenza vaccine ,Detergents ,Orthomyxoviridae ,medicine.disease_cause ,Sensitivity and Specificity ,Biochemistry ,High-performance liquid chromatography ,Analytical Chemistry ,Adsorption ,Influenza A virus ,medicine ,Solubility ,Chromatography, High Pressure Liquid ,Chromatography ,biology ,Chemistry ,Organic Chemistry ,Reproducibility of Results ,General Medicine ,Reversed-phase chromatography ,Silicon Dioxide ,biology.organism_classification ,Vaccines, Inactivated ,Influenza Vaccines ,Selectivity - Abstract
The characterization of influenza vaccine composition has been approached through a novel methodology suitable for routine analysis. It is based on a two-stage process involving an initial sample processing step followed by analysis by reversed-phase HPLC with UV detection. The sample processing involves an initial concentration step carried out in the presence of a combination of detergents and organic solvents to enhance solubilization and ultimately to provide adequate detection. Conditions that provided fast, reproducible and selective separations of vaccine constituents were investigated by reversed-phase HPLC. The use of non-porous silica stationary phases was found to minimize carry-over and non-specific adsorption observed with conventional columns. An evaluation of separation parameters, including mobile phase composition and column temperature, allowed optimization of the selectivity of the method. The optimized method was suitable for the characterization of processed monovalent preparations (containing influenza virus constituents from a single strain). In addition, it allowed the simultaneous detection of the three influenza subtypes in trivalent vaccines in a single analysis. Several influenza constituents were detected including nucleoprotein, the highly hydrophobic matrix protein and the primary surface antigen, haemagglutinin (HA).
- Published
- 2006
18. Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa
- Author
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Anita Kane, Grant E. Frahm, Daryl G.S. Smith, Michel Girard, Terry D. Cyr, Michael V. Johnston, and Barry Lorbetskie
- Subjects
Protein Structure Comparison ,Proteomics ,Protein Structure ,Glycosylation ,Science ,Lysine ,Size-exclusion chromatography ,Saccharomyces cerevisiae ,Biochemistry ,Peptide Mapping ,Mass Spectrometry ,Pichia ,Pichia pastoris ,law.invention ,chemistry.chemical_compound ,Glycation ,law ,Albumins ,medicine ,Humans ,Post-Translational Modification ,Serum Albumin ,Genetics ,Multidisciplinary ,biology ,Circular Dichroism ,Albumin ,Biology and Life Sciences ,Proteins ,Oryza ,biology.organism_classification ,Human serum albumin ,Plants, Genetically Modified ,Recombinant Proteins ,chemistry ,Recombinant DNA ,Medicine ,medicine.drug ,Research Article - Abstract
The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.
- Published
- 2014
19. Optimization and qualification of a quantitative reversed-phase HPLC method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays
- Author
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Caroline Gravel, Mike Walsh, Aline Rinfret, Michel Girard, Barry Lorbetskie, Xuguang Li, Jun Wang, and Cynthia Allen
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Radial immunodiffusion ,Detection limit ,Chromatography ,General Veterinary ,General Immunology and Microbiology ,Influenza vaccine ,Blotting, Western ,Public Health, Environmental and Occupational Health ,Reproducibility of Results ,Hemagglutinin Glycoproteins, Influenza Virus ,Reversed-phase chromatography ,Repeatability ,Hemagglutinin ,Biology ,Virology ,High-performance liquid chromatography ,Infectious Diseases ,Influenza A Virus, H1N1 Subtype ,Influenza Vaccines ,Limit of Detection ,Molecular Medicine ,Quantitative analysis (chemistry) ,Chromatography, High Pressure Liquid - Abstract
A previously described reversed-phase HPLC (RP-HPLC) method based on fast separations on a non-porous silica stationary phase [1] was optimized and qualified for the quantitative determination of hemagglutinin (HA) in influenza vaccine preparations. Optimization of the gradient elution conditions led to improved separation of the HA1 subunit from other vaccine constituents. The sensitivity of the method was significantly increased by using native fluorescence detection, resulting in an approximately 10-fold increase as compared to UV–vis detection. This enabled the elimination of the concentration step described in the original method and allowed direct analysis of vaccine preparations. The method was qualified for linearity, range, limit of detection, limit of quantitation and precision. Overall, it was found to be linear over the range of 2.5–100 μg HA/mL for all subtypes examined. This range covered 50–150% of the concentration found for individual strains in seasonal influenza vaccines and in the pandemic H1N1 vaccine. The limit of detection and limit of quantitation for each subtype were found to be suitable for the method's intended purpose and compared well to values found by the single radial immunodiffusion (SRID). The repeatability of the method gave RSD values below 5% for both retention time and peak areas. As expected for intermediate precision, larger RSD values for peak area were obtained but were below 10% and deemed acceptable. The RP-HPLC results were compared to Western blot analysis using a HA universal antibody for a set of 15 monovalent A/California H1N1 preparations and showed good correlation. Similarly, the quantitative nature of the RP-HPLC method was assessed in relation to the SRID assay currently used for the determination of the HA content in bulk antigen and final vaccine preparations. Thus, for a series of 23 monovalent A/Brisbane/59/2007 H1N1 bulks, ranging between 12.7 and 15.9 μg HA/mL by SRID, the RP-HPLC values were found to be in very good agreement, ranging between 11.9 and 14.1 μg HA/mL (n = 5) for five determinations carried out on 5 different days. During the 2009–10 H1N1 influenza pandemic the quantitative RP-HPLC method was used alongside several other test methods for the analysis of pandemic H1N1 vaccine preparations that included bulk antigen and final vaccines. The HA content of vaccines formulated at 15 or 30 μg/mL was measured by RP-HPLC and SRID and results showed that the HA content determined by RP-HPLC correlated well to that determined by SRID and to values determined by Western blot. Overall, the results provided further evidence of the usefulness of RP-HPLC for the detection and quantitation of the HA content once a reference standard has been established.
- Published
- 2010
20. Data set for mass spectrometric analysis of recombinant human serum albumin from various expression systems
- Author
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Daryl G.S. Smith, Grant E. Frahm, Terry D. Cyr, Michael V. Johnston, Michel Girard, Barry Lorbetskie, and Anita Kane
- Subjects
Arginine ,Lysine ,Oryza sativa ,lcsh:Computer applications to medicine. Medical informatics ,law.invention ,Glycation ,law ,medicine ,Hexose ,lcsh:Science (General) ,Data Article ,chemistry.chemical_classification ,Recombinant proteins ,Multidisciplinary ,Mass spectrometry ,Albumin ,Human serum albumin ,Yeast ,chemistry ,Biochemistry ,Recombinant DNA ,lcsh:R858-859.7 ,medicine.drug ,lcsh:Q1-390 - Abstract
Human serum albumin (HSA) is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3) (2012) 209–290). Due to the potential transmission of pathogens from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA) (Chen et al., Biochim. Biophys. Acta (BBA)—Gen. Subj. 1830(12) (2013) 5515–5525; Kobayashi, Biologicals 34(1) (2006) 55–59). Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15) (2012) 4661–4670), both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9) (2014) e109893). We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.
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