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In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed.

The methylotrophic yeast Pichia pastoris is widely used as a microbial host for recombinant protein production. Bioreactor models for P. pastoris can inform understanding of cellular metabolism and can be used to optimize bioreactor operation. This article constructs an extensive macroscopic bioreactor model for P. pastoris which describes substrates, biomass, total protein, other medium components, and off-gas components. Species and elemental balances are introduced to describe uptake and evolution rates for medium components and off-gas components. Additionally, a pH model is constructed using an overall charge balance, acid/base equilibria, and activity coefficients to describe production of recombinant protein and precipitation of medium components. The extent of run-to-run variability is modeled by distributions of a subset of the model parameters, which are estimated using the maximum likelihood method. Model prediction from the extensive macroscopic bioreactor model well describes experimental data with different operating conditions. The probability distributions of the model predictions quantified from the parameter distribution are quantifiably consistent with the run-to-run variability observed in the experimental data. The uncertainty description in this macroscopic bioreactor model identifies the model parameters that have large variability and provides guidance as to which aspects of cellular metabolism should be the focus of additional experimental studies. The model for medium components with pH and precipitation can be used for improving chemically defined medium by minimizing the amount of components needed while meeting cellular requirements.

Lentiviral vectors (LVs) are used in cell and gene therapies due to their ability to transduce both dividing and non-dividing cells while carrying a relatively large genetic payload and providing long-term gene expression via gene integration. Current cultivation methods produce titers of 105–107 transduction unit (TU)/mL; thus, it is necessary to concentrate LVs as well as remove process- and product-related impurities. In this work, we used a packaging cell line WinPac-RD-HV for LV production to simplify upstream processing. A direct capture method based on ion-exchange chromatography and cellulose nanofibers for LV concentration and purification was developed. This novel scalable stationary phase provides a high surface area that is accessible to LV and, therefore, has potential for high-capacity operation compared to traditional bead-based supports. We were able to concentrate LVs 100-fold while achieving a two-log removal of host cell protein and maintaining up to a 90% yield of functional vector., Graphical Abstract

Background: Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. Results: We describe a holistic approach for the molecular design of recombinant protein antigens—considering both their manufacturability and antigenicity—informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. Conclusions: This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits. Instituto de Virología Fil: Dalvie, Neil C. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Dalvie, Neil C. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Brady, Joseph R. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Brady, Joseph R. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Crowell, Laura E. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Crowell, Laura E. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Tracey, Mary Kate. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Biedermann, Andrew M. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Biedermann, Andrew M. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Kaur, Kawaljit. University of Kansas. Vaccine Analytics and Formulation Center. Department of Pharmaceutical Chemistry; Estados Unidos Fil: Hickey, John M. University of Kansas. Vaccine Analytics and Formulation Center. Department of Pharmaceutical Chemistry; Estados Unidos Fil: Kristensen II, D. Lee. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Bonnyman, Alexandra D. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Bonnyman, Alexandra D. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Rodriguez-Aponte, Sergio A. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Rodriguez-Aponte, Sergio A. Massachusetts Institute of Technology. Department of Biological Engineering; Estados Unidos Fil: Whittaker, Charles A. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Bok, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vega, Celina Guadalupe. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Vega, Celina Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mukhopadhyay, Tarit K. University College London. Department of Biochemical Engineering; Reino Unidos Fil: Joshi, Sangeeta B. University of Kansas. Vaccine Analytics and Formulation Center. Department of Pharmaceutical Chemistry; Estados Unidos Fil: Volkin, David B. University of Kansas. Vaccine Analytics and Formulation Center. Department of Pharmaceutical Chemistry; Estados Unidos Fil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Love, Kerry R. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Love, Kerry R. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos Fil: Love, J. Christopher. Massachusetts Institute of Technology. Department of Chemical Engineering; Estados Unidos Fil: Love, J. Christopher. Massachusetts Institute of Technology. The Koch Institute for Integrative Cancer Research; Estados Unidos

BackgroundVaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. ResultsWe describe a holistic approach for the molecular design of recombinant protein antigens—considering both their manufacturability and antigenicity—informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. ConclusionsThis study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.

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.

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein‐based rotavirus vaccine candidate derived from non‐replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small‐scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full‐length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full‐length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).

Depth filtration is employed in the production of many biological products. Although high throughput process development is available for most purification operations, options for high throughput depth filtration are limited. In this report, we describe the development and qualification of an ultra scale-down (USD) device for high throughput depth filtration. The system enables the parallel evaluation of eight single- or multi-layer depth filters approximately 0.2 cm2 in cross-sectional area, greater than two logs smaller than the smallest commercially available devices. To assess precision, scalability, modularity, and media heterogeneity in USD depth filtration, several types of adsorbent media were qualified in the USD device, with filtration performance compared to lab-scale devices (cross-sectional area: 23–25 cm2). The performance was further investigated with several high throughput clarity measurements, including particle characterization of filtrate fractions. Historically, depth filtration development has been limited by the amount of material required to run lab-scale filters. The described high throughput technology enables the screening of filter media early in development, optimization of operating parameters, and coupling of upstream operations to filtration performance. These high throughput capabilities will create efficiencies for development and manufacturing that were unattainable previously and create an important tool for screening novel biological entities.

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.

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. © 2015 Wiley Periodicals, Inc.

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018.

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.

Determination of copy number for integrated expression units in selected P. pastoris clones. Description of the methodology used for quantification of copy number for expression cassettes integrated in selected P. pastoris clones. The method is based on real time PCR amplification of zeocin gene. (DOCX 186 kb)

Serum IgG reactivity against the â emptyâ K1-K1 HBc VLPs and against irrelevant antigens (Cytomegalovirus antigen, purified EBV capsid protein, Toxoplasma gondii antigen). Reactivity of mouse sera with carrier HBc-derived VLPs (positive control) in comparison with irrelevant antigens (negative control) demonstrating specificity of ELISA method used. (PPTX 91 kb)

Aim: The aim of this study was to investigate the role of quorum sensing in Bacillus anthracis growth and toxin production. Methods and Results: A microwell plate culture method was developed to simulate the normal UK-licensed anthrax vaccine production run. Once established, sterile supernatant additions from a previous B. anthracis culture were made, and reductions in lag phase and early stimulation of the anthrax toxin component protective antigen (PA) were monitored using ELISA. The addition of the quorum-sensing inhibitor, fur-1, prolonged the lag phase and impeded PA production. Spin filters of various sizes were used to identify the molecular weight fraction of the sterile supernatant responsible for the autoinducer effect. A weight fraction between 5 and 10 kDa was responsible for the autoinducer effect; however, further identification using mass spectroscopy proved inconclusive. Conclusions: Quorum sensing mediated by the autoinducer two molecule plays a significant role in both B. anthracis growth and toxin production. Significance and Impact of the study: While genomic analysis has eluded to the importance of LuxS and quorum sensing in anthrax, this is the first analysis using a production strain of B. anthracis and a quorum-sensing inhibitor to monitor the effect on growth and toxin production. This gives insights into anthrax pathogenicity and vaccine manufacture.

The licensed UK anthrax vaccine is produced by static cultures of Bacillus anthracis Sterne 34F2 in glass Thompson bottles, each batch consisting of multiple bottles grown for 24–28 h. In this work, a novel miniature bioreactor was used as a scale-down tool to investigate the possible transfer of anthrax vaccine production from static culture to stirred tank operation and to explore the effects of this change in culture conditions on process performance. It is shown that the change to stirred culture conditions is possible and that the concentration of the two main vaccine components, Protective Antigen (PA) and Lethal Factor (LF), are reached in less than half the time compared to standard Thompson bottle methods. Furthermore, because higher cell densities were attained in the miniature bioreactor, a 74% increase in antigen concentration was achieved. More detailed analysis of the stirred bioreactor results operated with and without aeration showed antigen degradation in the presence of aeration. Overall this work demonstrates the usefulness of miniaturisation techniques to perform process characterisation studies safely and without significant capital investment for large-scale containment.

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.

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.

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.

ProSavin(®) is a lentiviral vector (LV)-based gene therapy for Parkinson's disease. ProSavin(®) is currently in a Phase I/II clinical trial using material that was generated by transient transfection of adherent human embryonic kidney (HEK)293T cells. For future large-scale productions of ProSavin(®), we have previously reported the development and characterization of two inducible producer cell lines, termed PS5.8 and PS46.2. PS46.2 has been successfully adapted to grow in suspension cultures. The present study describes the creation of a small-scale (

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.

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.

Serum IgG reactivity against the â emptyâ K1-K1 HBc VLPs and against irrelevant antigens (Cytomegalovirus antigen, purified EBV capsid protein, Toxoplasma gondii antigen). Reactivity of mouse sera with carrier HBc-derived VLPs (positive control) in comparison with irrelevant antigens (negative control) demonstrating specificity of ELISA method used. (PPTX 91 kb)

Determination of copy number for integrated expression units in selected P. pastoris clones. Description of the methodology used for quantification of copy number for expression cassettes integrated in selected P. pastoris clones. The method is based on real time PCR amplification of zeocin gene. (DOCX 186 kb)