7 research outputs found
On-Line influenza virus quantification for viral production processes thanks to affinity-based surface plasmon resonance biosensor
Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. Recent progress made regarding cell culture bioprocesses allowed for numerous alternative strategies to developed future vaccine candidates, as for example the recombinant HA or Virusâlike Particles (VLP) vaccines. However, while cell culture allows for more versatility than ovoculture, regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. To drive these developments, WHO and regulatory agencies underlined the need for developing better influenza vaccine potency assays1,2. Actual influenza vaccine formulation and lot release rely on single-radial immunodiffusion (SRID) assay, which requires strain-specific reference sera and antigen reagents. However, the annual preparation of these reagents takes between 2 to 6 months and constitutes a critical bottleneck for the release of vaccine lots3. Additionally, SRID is not implementable for process development as such technique cannot handle in-process low concentrated and non-purified material. We developed an assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigen and influenza virus based on surface plasmon resonance (SPR). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans present at the surface of the fetuin-functionalized sensor. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses on a unique sensor. Two types of purified standard were used during the development of the assay. Commercial trivalent inactivated vaccine (âTIVâ) has been used for the determination of optimal analytical conditions, while a stock of split inactivated H1N1 virus has been produced and calibrated in our laboratory to study the specific response obtained toward this HA subtype. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng/mL-20 ”g/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, and a better reproducibility than SRID and its potential alternatives recently proposed (1,4,5. Additionally, the applicability of this assay for an on-line vaccine production monitoring has been validated by off-line measurement of influenza H1N1 virus particles derived from cell culture supernatant. Such a test allowed to achieve a LOD of 106 Infectious Viral Particles/mL Thus, our assay provides an innovative tool to evaluate influenza new vaccine bioprocesses, from viral production kinetics in mammalian cell culture to vaccine potency evaluation
Développement d'une nouvelle technique bioanalytique basée sur l'imagerie par résonance plasmonique de surface pour le suivi en-ligne de virus influenza dans les bioprocédés vaccinaux
Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. However, while cell culture allows for more versatility regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. FDAâs Process analytical Technology (PAT) and Quality by Design (QbD) guidances impels vaccine manufacturers to implement on-line technologies to monitor both critical process parameters (CPP) and the bioproductâs critical quality attributes (CQA) within bioprocesses. Today, Influenza vaccine potency is evaluated by single-radial immunodiffusion (SRID), which requires the annual production of strain-specific reagents that constitutes a potential bottleneck for the release of vaccine lots and cannot be implemented for in-process monitoring. On the other hand, while recent physical quantification techniques appear more relevant than traditional infectivity assays for influenza virus monitoring as the ratio of total to infectious influenza virus vary from 10-1000, they are not potency-indicating. During this thesis, we developed and evaluated a potency-indicating assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigens and whole influenza viruses based on quantitative surface plasmon resonance imaging (SPRi). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses. Influenza vaccine and virus samples derived from various cell culture platforms were processed onto the sensor to evaluate viral production kinetics. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng HA/mL-20 ÎŒg HA/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, as well as a better reproducibility than SRID and its potential alternatives (<5% RSD vs 6-15%). Such a test also allowed to achieve a LOD <106 Infectious Viral Particles/mL. The applicability of this assay for in-process monitoring of influenza vaccine production has been validated by the determination of influenza virus production kinetics in good correlation with both total viral particle content and total HA protein content in two production cell lines. We validated the potency-indicating nature of this assay. Finally, we designed and evaluated an automated sampling and purification line for at-line (frequency of analysis : 1h-1) monitoring of bioactive influenza virus production kinetics in suspension cell culture. Thus, this work provides an innovative tool for the development of novel influenza vaccine bioprocesses, supporting the PAT initiativeLes Ă©pidĂ©mies saisonniĂšres du virus de la grippe, associĂ©es Ă la menace constante d'une nouvelle pandĂ©mie, incitent les fabricants de vaccins Ă dĂ©velopper des procĂ©dĂ©s plus rĂ©actifs que le procĂ©dĂ© traditionnel basĂ© sur lâovoculture. Bien que les procĂ©dĂ©s alternatifs dĂ©veloppĂ©s, basĂ©s sur la culture cellulaire, offrent de nombreux avantages ils nĂ©cessitent encre des optimisations afin de pouvoir concurrencer sĂ©rieusement le processus traditionnel. Ainsi, les autoritĂ©s rĂ©gulatrices incitent les fabricants de vaccins Ă dĂ©velopper des outils analytiques en-ligne pour surveiller Ă la fois les paramĂštres critiques du procĂ©dĂ© (CPP) et les attributs de qualitĂ© critiques (CQA) des produits dans les bioprocĂ©dĂ©s dans le cadre de la dĂ©marche Process Analytical Technology. Aujourd'hui, la technique de rĂ©fĂ©rence pour le dosage dâactivitĂ© vaccinale est lâimmunodiffusion radiale (SRID). Elle nĂ©cessite une production annuelle de rĂ©actifs potentiellement critique pour la libĂ©ration des lots et ne peut pas ĂȘtre mis en Ćuvre pour du suivi de procĂ©dĂ©. Les techniques de quantification physique (TRPS, viromĂ©trie, NTAâŠ) rĂ©centes apparaissent plus appropriĂ©es que les dosages d'infectivitĂ© traditionnels pour du suivi en-ligne Ă©tant donnĂ© le ratio entre les particules virales totales et infectieuse de 10-10000. Cependant, ces techniques ne fournissent pas dâinformation concernant lâactivitĂ© vaccinale. Au cours de cette thĂšse a Ă©tĂ© dĂ©veloppĂ© un test analytique pour la quantification de l'hĂ©magglutinine (HA) et de virus bioactifs basĂ© sur l'imagerie par rĂ©sonance plasmonique de surface (SPRi). La mĂ©thode est basĂ©e sur la capture par affinitĂ© de HA par les glycanes possĂ©dant une extrĂ©mitĂ© terminale de type acide sialique. Les conditions ont Ă©tĂ© optimisĂ©es pour offrir une rĂ©gĂ©nĂ©ration de la surface afin d'effectuer des analyses sĂ©quentielles. Des Ă©chantillons de vaccins et de virus produits sur diverses plateformes/substrats de culture cellulaire ont Ă©tĂ© analysĂ©s sur le capteur dans le but dâĂ©valuer les cinĂ©tiques de production virale. Ce test permet une Ă©valuation rapide (<10 mins) de lâactivitĂ© de l'hĂ©magglutinine grippale avec une gamme analytique large (30 ng HA.mL-1-20 ÎŒg HA.mL-1). De plus, la technique offre une limite de dĂ©tection (LOD) 100 fois infĂ©rieure Ă SRID, ainsi qu'une meilleure reproductibilitĂ© que la SRID et ses alternatives potentielles (<5% RSD vs 6-15%). Un tel test a Ă©galement permis d'atteindre une LOD <106 particules virales infectieuses.mL-1. L'applicabilitĂ© de ce test pour le suivi en-ligne des bioprocĂ©dĂ©s a Ă©tĂ© validĂ©e par lâĂ©valuations de cinĂ©tiques de production virales, offrant bonne corrĂ©lation avec la concentration en particules virales totales et la quantitĂ© totale en protĂ©ines HA dans deux lignĂ©es cellulaires de production. Nous avons Ă©galement validĂ© que ce test permettait une Ă©valuation de lâactivitĂ© pour du virus entier. Enfin, nous avons conçu et Ă©valuĂ© une ligne d'Ă©chantillonnage et de purification automatisĂ©e pour lâĂ©valuation en-ligne de la cinĂ©tique de production virale dans une culture cellulaire aviaire en suspension. Ce travail de thĂšse a ainsi permis de fournir un outil innovant pour le dĂ©veloppement de nouveaux bioprocĂ©dĂ©s vaccinaux (suivi en-ligne, formulation, Ă©tudes de stabilitĂ©), soutenant l'initiative PA
Développement d'une nouvelle technique bioanalytique basée sur l'imagerie par résonance plasmonique de surface pour le suivi en-ligne de virus influenza dans les bioprocédés vaccinaux
Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. However, while cell culture allows for more versatility regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. FDAâs Process analytical Technology (PAT) and Quality by Design (QbD) guidances impels vaccine manufacturers to implement on-line technologies to monitor both critical process parameters (CPP) and the bioproductâs critical quality attributes (CQA) within bioprocesses. Today, Influenza vaccine potency is evaluated by single-radial immunodiffusion (SRID), which requires the annual production of strain-specific reagents that constitutes a potential bottleneck for the release of vaccine lots and cannot be implemented for in-process monitoring. On the other hand, while recent physical quantification techniques appear more relevant than traditional infectivity assays for influenza virus monitoring as the ratio of total to infectious influenza virus vary from 10-1000, they are not potency-indicating. During this thesis, we developed and evaluated a potency-indicating assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigens and whole influenza viruses based on quantitative surface plasmon resonance imaging (SPRi). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses. Influenza vaccine and virus samples derived from various cell culture platforms were processed onto the sensor to evaluate viral production kinetics. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng HA/mL-20 ÎŒg HA/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, as well as a better reproducibility than SRID and its potential alternatives (<5% RSD vs 6-15%). Such a test also allowed to achieve a LOD <106 Infectious Viral Particles/mL. The applicability of this assay for in-process monitoring of influenza vaccine production has been validated by the determination of influenza virus production kinetics in good correlation with both total viral particle content and total HA protein content in two production cell lines. We validated the potency-indicating nature of this assay. Finally, we designed and evaluated an automated sampling and purification line for at-line (frequency of analysis : 1h-1) monitoring of bioactive influenza virus production kinetics in suspension cell culture. Thus, this work provides an innovative tool for the development of novel influenza vaccine bioprocesses, supporting the PAT initiativeLes Ă©pidĂ©mies saisonniĂšres du virus de la grippe, associĂ©es Ă la menace constante d'une nouvelle pandĂ©mie, incitent les fabricants de vaccins Ă dĂ©velopper des procĂ©dĂ©s plus rĂ©actifs que le procĂ©dĂ© traditionnel basĂ© sur lâovoculture. Bien que les procĂ©dĂ©s alternatifs dĂ©veloppĂ©s, basĂ©s sur la culture cellulaire, offrent de nombreux avantages ils nĂ©cessitent encre des optimisations afin de pouvoir concurrencer sĂ©rieusement le processus traditionnel. Ainsi, les autoritĂ©s rĂ©gulatrices incitent les fabricants de vaccins Ă dĂ©velopper des outils analytiques en-ligne pour surveiller Ă la fois les paramĂštres critiques du procĂ©dĂ© (CPP) et les attributs de qualitĂ© critiques (CQA) des produits dans les bioprocĂ©dĂ©s dans le cadre de la dĂ©marche Process Analytical Technology. Aujourd'hui, la technique de rĂ©fĂ©rence pour le dosage dâactivitĂ© vaccinale est lâimmunodiffusion radiale (SRID). Elle nĂ©cessite une production annuelle de rĂ©actifs potentiellement critique pour la libĂ©ration des lots et ne peut pas ĂȘtre mis en Ćuvre pour du suivi de procĂ©dĂ©. Les techniques de quantification physique (TRPS, viromĂ©trie, NTAâŠ) rĂ©centes apparaissent plus appropriĂ©es que les dosages d'infectivitĂ© traditionnels pour du suivi en-ligne Ă©tant donnĂ© le ratio entre les particules virales totales et infectieuse de 10-10000. Cependant, ces techniques ne fournissent pas dâinformation concernant lâactivitĂ© vaccinale. Au cours de cette thĂšse a Ă©tĂ© dĂ©veloppĂ© un test analytique pour la quantification de l'hĂ©magglutinine (HA) et de virus bioactifs basĂ© sur l'imagerie par rĂ©sonance plasmonique de surface (SPRi). La mĂ©thode est basĂ©e sur la capture par affinitĂ© de HA par les glycanes possĂ©dant une extrĂ©mitĂ© terminale de type acide sialique. Les conditions ont Ă©tĂ© optimisĂ©es pour offrir une rĂ©gĂ©nĂ©ration de la surface afin d'effectuer des analyses sĂ©quentielles. Des Ă©chantillons de vaccins et de virus produits sur diverses plateformes/substrats de culture cellulaire ont Ă©tĂ© analysĂ©s sur le capteur dans le but dâĂ©valuer les cinĂ©tiques de production virale. Ce test permet une Ă©valuation rapide (<10 mins) de lâactivitĂ© de l'hĂ©magglutinine grippale avec une gamme analytique large (30 ng HA.mL-1-20 ÎŒg HA.mL-1). De plus, la technique offre une limite de dĂ©tection (LOD) 100 fois infĂ©rieure Ă SRID, ainsi qu'une meilleure reproductibilitĂ© que la SRID et ses alternatives potentielles (<5% RSD vs 6-15%). Un tel test a Ă©galement permis d'atteindre une LOD <106 particules virales infectieuses.mL-1. L'applicabilitĂ© de ce test pour le suivi en-ligne des bioprocĂ©dĂ©s a Ă©tĂ© validĂ©e par lâĂ©valuations de cinĂ©tiques de production virales, offrant bonne corrĂ©lation avec la concentration en particules virales totales et la quantitĂ© totale en protĂ©ines HA dans deux lignĂ©es cellulaires de production. Nous avons Ă©galement validĂ© que ce test permettait une Ă©valuation de lâactivitĂ© pour du virus entier. Enfin, nous avons conçu et Ă©valuĂ© une ligne d'Ă©chantillonnage et de purification automatisĂ©e pour lâĂ©valuation en-ligne de la cinĂ©tique de production virale dans une culture cellulaire aviaire en suspension. Ce travail de thĂšse a ainsi permis de fournir un outil innovant pour le dĂ©veloppement de nouveaux bioprocĂ©dĂ©s vaccinaux (suivi en-ligne, formulation, Ă©tudes de stabilitĂ©), soutenant l'initiative PA
Development of a new bioanalytical technique based on surface plasmon resonance imaging for the on-line monitoring of influenza viruses influenza viruses in vaccine bioprocesses
Les Ă©pidĂ©mies saisonniĂšres du virus de la grippe, associĂ©es Ă la menace constante d'une nouvelle pandĂ©mie, incitent les fabricants de vaccins Ă dĂ©velopper des procĂ©dĂ©s plus rĂ©actifs que le procĂ©dĂ© traditionnel basĂ© sur lâovoculture. Bien que les procĂ©dĂ©s alternatifs dĂ©veloppĂ©s, basĂ©s sur la culture cellulaire, offrent de nombreux avantages ils nĂ©cessitent encre des optimisations afin de pouvoir concurrencer sĂ©rieusement le processus traditionnel. Ainsi, les autoritĂ©s rĂ©gulatrices incitent les fabricants de vaccins Ă dĂ©velopper des outils analytiques en-ligne pour surveiller Ă la fois les paramĂštres critiques du procĂ©dĂ© (CPP) et les attributs de qualitĂ© critiques (CQA) des produits dans les bioprocĂ©dĂ©s dans le cadre de la dĂ©marche Process Analytical Technology. Aujourd'hui, la technique de rĂ©fĂ©rence pour le dosage dâactivitĂ© vaccinale est lâimmunodiffusion radiale (SRID). Elle nĂ©cessite une production annuelle de rĂ©actifs potentiellement critique pour la libĂ©ration des lots et ne peut pas ĂȘtre mis en Ćuvre pour du suivi de procĂ©dĂ©. Les techniques de quantification physique (TRPS, viromĂ©trie, NTAâŠ) rĂ©centes apparaissent plus appropriĂ©es que les dosages d'infectivitĂ© traditionnels pour du suivi en-ligne Ă©tant donnĂ© le ratio entre les particules virales totales et infectieuse de 10-10000. Cependant, ces techniques ne fournissent pas dâinformation concernant lâactivitĂ© vaccinale. Au cours de cette thĂšse a Ă©tĂ© dĂ©veloppĂ© un test analytique pour la quantification de l'hĂ©magglutinine (HA) et de virus bioactifs basĂ© sur l'imagerie par rĂ©sonance plasmonique de surface (SPRi). La mĂ©thode est basĂ©e sur la capture par affinitĂ© de HA par les glycanes possĂ©dant une extrĂ©mitĂ© terminale de type acide sialique. Les conditions ont Ă©tĂ© optimisĂ©es pour offrir une rĂ©gĂ©nĂ©ration de la surface afin d'effectuer des analyses sĂ©quentielles. Des Ă©chantillons de vaccins et de virus produits sur diverses plateformes/substrats de culture cellulaire ont Ă©tĂ© analysĂ©s sur le capteur dans le but dâĂ©valuer les cinĂ©tiques de production virale. Ce test permet une Ă©valuation rapide (<10 mins) de lâactivitĂ© de l'hĂ©magglutinine grippale avec une gamme analytique large (30 ng HA.mL-1-20 ÎŒg HA.mL-1). De plus, la technique offre une limite de dĂ©tection (LOD) 100 fois infĂ©rieure Ă SRID, ainsi qu'une meilleure reproductibilitĂ© que la SRID et ses alternatives potentielles (<5% RSD vs 6-15%). Un tel test a Ă©galement permis d'atteindre une LOD <106 particules virales infectieuses.mL-1. L'applicabilitĂ© de ce test pour le suivi en-ligne des bioprocĂ©dĂ©s a Ă©tĂ© validĂ©e par lâĂ©valuations de cinĂ©tiques de production virales, offrant bonne corrĂ©lation avec la concentration en particules virales totales et la quantitĂ© totale en protĂ©ines HA dans deux lignĂ©es cellulaires de production. Nous avons Ă©galement validĂ© que ce test permettait une Ă©valuation de lâactivitĂ© pour du virus entier. Enfin, nous avons conçu et Ă©valuĂ© une ligne d'Ă©chantillonnage et de purification automatisĂ©e pour lâĂ©valuation en-ligne de la cinĂ©tique de production virale dans une culture cellulaire aviaire en suspension. Ce travail de thĂšse a ainsi permis de fournir un outil innovant pour le dĂ©veloppement de nouveaux bioprocĂ©dĂ©s vaccinaux (suivi en-ligne, formulation, Ă©tudes de stabilitĂ©), soutenant l'initiative PATInfluenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. However, while cell culture allows for more versatility regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. FDAâs Process analytical Technology (PAT) and Quality by Design (QbD) guidances impels vaccine manufacturers to implement on-line technologies to monitor both critical process parameters (CPP) and the bioproductâs critical quality attributes (CQA) within bioprocesses. Today, Influenza vaccine potency is evaluated by single-radial immunodiffusion (SRID), which requires the annual production of strain-specific reagents that constitutes a potential bottleneck for the release of vaccine lots and cannot be implemented for in-process monitoring. On the other hand, while recent physical quantification techniques appear more relevant than traditional infectivity assays for influenza virus monitoring as the ratio of total to infectious influenza virus vary from 10-1000, they are not potency-indicating. During this thesis, we developed and evaluated a potency-indicating assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigens and whole influenza viruses based on quantitative surface plasmon resonance imaging (SPRi). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses. Influenza vaccine and virus samples derived from various cell culture platforms were processed onto the sensor to evaluate viral production kinetics. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng HA/mL-20 ÎŒg HA/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, as well as a better reproducibility than SRID and its potential alternatives (<5% RSD vs 6-15%). Such a test also allowed to achieve a LOD <106 Infectious Viral Particles/mL. The applicability of this assay for in-process monitoring of influenza vaccine production has been validated by the determination of influenza virus production kinetics in good correlation with both total viral particle content and total HA protein content in two production cell lines. We validated the potency-indicating nature of this assay. Finally, we designed and evaluated an automated sampling and purification line for at-line (frequency of analysis : 1h-1) monitoring of bioactive influenza virus production kinetics in suspension cell culture. Thus, this work provides an innovative tool for the development of novel influenza vaccine bioprocesses, supporting the PAT initiativ
SPRi-based hemagglutinin quantitative assay for influenza vaccine production monitoring
International audienc
High Risk of Anal and Rectal Cancer in Patients With Anal and/or Perianal Crohnâs Disease
International audienceBackground & AimsLittle is known about the magnitude of the risk of anal and rectal cancer in patients with anal and/or perineal Crohnâs disease. We aimed to assess the risk of anal and rectal cancer in patients with Crohnâs perianal disease followed up in the Cancers Et Surrisque AssociĂ© aux Maladies Inflammatoires Intestinales En France (CESAME) cohort.MethodsWe collected data from 19,486 patients with inflammatory bowel disease (IBD) enrolled in the observational CESAME study in France, from May 2004 through June 2005; 14.9% of participants had past or current anal and/or perianal Crohnâs disease. Subjects were followed up for a median time of 35 months (interquartile range, 29â40 mo). To identify risk factors for anal cancer in the total CESAME population, we performed a case-control study in which participants were matched for age and sex.ResultsAmong the total IBD population, 8 patients developed anal cancer and 14 patients developed rectal cancer. In the subgroup of 2911 patients with past or current anal and/or perianal Crohnâs lesions at cohort entry, 2 developed anal squamous-cell carcinoma, 3 developed perianal fistulaârelated adenocarcinoma, and 6 developed rectal cancer. The corresponding incidence rates were 0.26 per 1000 patient-years for anal squamous-cell carcinoma, 0.38 per 1000 patient-years for perianal fistulaârelated adenocarcinoma, and 0.77 per 1000 patient-years for rectal cancer. Among the 16,575 patients with ulcerative colitis or Crohnâs disease without anal or perianal lesions, the incidence rate of anal cancer was 0.08 per 1000 patient-years and of rectal cancer was 0.21 per 1000 patient-years. Among factors tested by univariate conditional regression (IBD subtype, disease duration, exposure to immune-suppressive therapy, presence of past or current anal and/or perianal lesions), the presence of past or current anal and/or perianal lesions at cohort entry was the only factor significantly associated with development of anal cancer (odds ratio, 11.2; 95% CI, 1.18-551.51; P = .03).ConclusionsIn an analysis of data from the CESAME cohort in France, patients with anal and/or perianal Crohnâs disease have a high risk of anal cancer, including perianal fistulaârelated cancer, and a high risk of rectal cancer