Tarit Mukhopadhyay

Rapid characterization of outer-membrane proteins in Neisseria lactamica by SELDI-TOF-MS (surface-enhanced laser desorption ionization-time-of-flight MS) for use in a meningococcal vaccine.

Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal Neisseria species, particularly with Neisseria lactamica. We have reported previously that immunization with N. lactamica outer‐membrane vesicles containing the major OMPs (outer‐membrane proteins) protected mice against lethal challenge with meningococci of diverse serogroups and serotypes and has the potential to form the basis of a vaccine against meningococcal diseases [Oliver, Reddin, Bracegirdle et al. (2002) Infect. Immun. 70, 3621–3626]. In the present study, we have shown that biomass production and the profile of outer‐membrane vesicle proteins may be affected by fermentation conditions and, in particular, media composition. Ciphergen SELDI–TOF Protein Chips® were used as a rapid and sensitive new method in comparison with conventional SDS/PAGE. SELDI–TOF‐MS (surface‐enhanced laser‐desorption ionization–time‐of‐flight MS) reproducibly identified three major OMPs (NspA, RmpM and PorB) and detected the changes in the protein profile when the growth medium was altered. The findings of this work indicate that SELDI–TOF‐MS is a useful tool for the rapid optimization of OMP production in industrial fermentation processes and can be adapted as a Process Analytical Technology (http://www.fda.gov/cder/OPS/PAT.htm).

Quantitative high throughput analytics to support polysaccharide production process development

The rapid development of purification processes for polysaccharide vaccines is constrained by a lack of analytical tools current technologies for the measurement of polysaccharide recovery and process-related impurity clearance are complex, time-consuming, and generally not amenable to high throughput process development (HTPD). HTPD is envisioned to be central to the improvement of existing polysaccharide manufacturing processes through the identification of critical process parameters that potentially impact the quality attributes of the vaccine and to the development of de novo processes for clinical candidates, across the spectrum of downstream processing. The availability of a fast and automated analytics platform will expand the scope, robustness, and evolution of Design of Experiment (DOE) studies. This paper details recent advances in improving the speed, throughput, and success of in-process analytics at the micro-scale. Two methods, based on modifications of existing procedures, are described for the rapid measurement of polysaccharide titre in microplates without the need for heating steps. A simplification of a commercial endotoxin assay is also described that features a single measurement at room temperature. These assays, along with existing assays for protein and nucleic acids are qualified for deployment in the high throughput screening of polysaccharide feedstreams. Assay accuracy, precision, robustness, interference, and ease of use are assessed and described. In combination, these assays are capable of measuring the product concentration and impurity profile of a microplate of 96 samples in less than one day. This body of work relies on the evaluation of a combination of commercially available and clinically relevant polysaccharides to ensure maximum versatility and reactivity of the final assay suite. Together, these advancements reduce overall process time by up to 30-fold and significantly reduce sample volume over current practices. The assays help build an analytical foundation to support the advent of HTPD technology for polysaccharide vaccines. It is envisaged that this will lead to an expanded use of Quality by Design (QbD) studies in vaccine process development.

High throughput screening of particle conditioning operations: I. System design and method development

The biotech industry is under increasing pressure to decrease both time to market and development costs. Simultaneously, regulators are expecting increased process understanding. High throughput process development (HTPD) employs small volumes, parallel processing, and high throughput analytics to reduce development costs and speed the development of novel therapeutics. As such, HTPD is increasingly viewed as integral to improving developmental productivity and deepening process understanding. Particle conditioning steps such as precipitation and flocculation may be used to aid the recovery and purification of biological products. In this first part of two articles, we describe an ultra scale‐down system (USD) for high throughput particle conditioning (HTPC) composed of off‐the‐shelf components. The apparatus is comprised of a temperature‐controlled microplate with magnetically driven stirrers and integrated with a Tecan liquid handling robot. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. A comprehensive suite of high throughput analytics enables measurement of product titer, product quality, impurity clearance, clarification efficiency, and particle characterization. HTPC at the 1 mL scale was evaluated with fermentation broth containing a vaccine polysaccharide. The response profile was compared with the Pilot‐scale performance of a non‐geometrically similar, 3 L reactor. An engineering characterization of the reactors and scale‐up context examines theoretical considerations for comparing this USD system with larger scale stirred reactors. In the second paper, we will explore application of this system to industrially relevant vaccines and test different scale‐up heuristics. Biotechnol. Bioeng. 2015;112: 1554–1567.

Production and purification of chimeric HBc virus-like particles carrying influenza virus LAH domain as vaccine candidates

BackgroundThe lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology.ResultsFermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice.ConclusionOur results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.

High throughput quantification of capsular polysaccharides for multivalent vaccines using precipitation with a cationic surfactant

The increasing requirement for multivalent vaccines containing diverse capsular polysaccharides has created an unmet need for a fast and straightforward assay for polysaccharide titer. We describe a novel and robust assay for the quantitation of anionic capsular polysaccharides. The binding of hexadecyltrimethyammonium bromide (Hb) to anionic capsular polysaccharides results in a precipitation reaction wherein the suspension turbidity is proportional to polysaccharide titer. The turbidity can be quickly measured as absorbance across a range of wavelengths that resolve scattering light. Carbohydrates comprised of repeating units of one to seven monosaccharides with phosphodiester groups, uronic acids, and sialic acids all reacted strongly and there does not appear to be specificity with respect to the particular anionic moiety. The assay is compatible with an array of common buffers across a pH range of 3.0-8.75 and with NaCl concentration exceeding 400 mM. Interference from DNA can be eliminated with a short incubation step with DNase. With these treatments, the assay has been employed in samples as complex as fermentation broth. A two-log dynamic range has been established with a mean relative standard deviation less than 10% across this range although inferior performance has been observed in fermentation broth. The precipitation assay enables the rapid quantitation of anionic polysaccharides. The resulting procedure can robustly measure the titer of myriad anionic capsular polysaccharides (CPS) in 96 samples in less than 30 min using low toxicity reagents and routine laboratory equipment. This development will greatly reduce the effort required to measure polysaccharide titer and yield during process development of polysaccharide vaccines.

Use of microwells to investigate the effect of quorum sensing on growth and antigen production in Bacillus anthracis Sterne 34F2

Aim:  The aim of this study was to investigate the role of quorum sensing in Bacillus anthracis growth and toxin production.

Vaccines Europe 2009.

The Informa Life Sciences vaccines conference is an annual meeting of a relatively small number of academics and industrialists. It is split into three concurrent sessions covering vaccine discovery, quality and manufacturing. Although there were many presentations of merit, only a few will be discussed here, including the plenary speeches on adjuvants and influenza.

High throughput screening of particle conditioning operations: II. Evaluation of scale‐up heuristics with prokaryotically expressed polysaccharide vaccines

Multivalent polysaccharide conjugate vaccines are typically comprised of several different polysaccharides produced with distinct and complex production processes. Particle conditioning steps, such as precipitation and flocculation, may be used to aid the recovery and purification of such microbial vaccine products. An ultra scale‐down approach to purify vaccine polysaccharides at the micro‐scale would greatly enhance productivity, robustness, and speed the development of novel conjugate vaccines. In part one of this series, we described a modular and high throughput approach to develop particle conditioning processes (HTPC) for biologicals that combines flocculation, solids removal, and streamlined analytics. In this second part of the series, we applied HTPC to industrially relevant feedstreams comprised of capsular polysaccharides (CPS) from several bacterial species. The scalability of HTPC was evaluated between 0.8 mL and 13 L scales, with several different scaling methodologies examined. Clarification, polysaccharide yield, impurity clearance, and product quality achieved with HTPC were reproducible and comparable with larger scales. Particle sizing was the response with greatest sensitivity to differences in processing scale and enabled the identification of useful scaling rules. Scaling with constant impeller tip speed or power per volume in the impeller swept zone offered the most accurate scale up, with evidence that time integration of these values provided the optimal basis for scaling. The capability to develop a process at the micro‐scale combined with evidence‐based scaling metrics provide a significant advance for purification process development of vaccine processes. The USD system offers similar opportunities for HTPC of proteins and other complex biological molecules. Biotechnol. Bioeng. 2015;112: 1568–1582.

Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes

Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136–370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

Ultra scale-down approaches to study the centrifugal harvest for viral vaccine production

Large scale continuous cell‐line cultures promise greater reproducibility and efficacy for the production of influenza vaccines, and adenovirus for gene therapy. This paper seeks to use an existing validated ultra scale‐down tool, which is designed to mimic the commercial scale process environment using only milliliters of material, to provide some initial insight into the performance of the harvest step for these processes. The performance of industrial scale centrifugation and subsequent downstream process units is significantly affected by shear. The properties of these cells, in particular their shear sensitivity, may be changed considerably by production of a viral product, but literature on this is limited to date. In addition, the scale‐down tool used here has not previously been applied to the clarification of virus production processes. The results indicate that virus infected cells do not actually show any increase in sensitivity to shear, and may indeed become less shear sensitive, in a similar manner to that previously observed in old or dead cell cultures. Clarification may be most significantly dependent on the virus release mechanism, with the budding influenza virus producing a much greater decrease in clarification than the lytic, non‐enveloped adenovirus. A good match was also demonstrated to the industrial scale performance in terms of clarification, protein release, and impurity profile.