A high-throughput single-use platform for vaccine bioprocess development

Vaccination is the predominant tool in the prevention of infectious disease. Considering the SARS-CoV-2 (Covid-19) pandemic, the need for a development platform, capable of rapid candidate screening and/or a vaccine scaffold capable of adaptability to new disease targets, has never been more apparent. VLPs, expressed in the methylotrophic yeast Pichia pastoris, offer an exciting alternative to current manufacturing methods due to their potential as scaffolds for foreign antigen display. The hepatitis B core antigen (HBC) can spontaneously self-assemble, forming icosahedral particles that are inherently immunogenic. Tandem Core HBC (TC-HBC) VLPs have been genetically modified in the major insertion region (MIR) enabling display of up to two epitopes of interest when assembled. For TC-HBC VLPs to be considered a viable vaccine candidate, their bioprocessing must be optimized. Currently, there are various issues to address, including problems with formation, solubility, and immunogenicity. Please click Additional Files below to see the full abstract

Ultra scale-down concepts to address early stage process development challenges in integrated continuous bioprocessing

The benefits of continuous bioprocessing, e.g. accelerated process development and scale-up, reduced capital costs, and standardisation, could be achieved through facility automation, universal process architecture, and the alignment of operational structures for the development and manufacturing organisations [1]. Both control strategy and rational design of universal process architecture demand an understanding of the limits and interdependence of these unit operations, and knowledge on how these could be controlled to sustain desired product quality over long periods of time. For example, to effectively implement global process control, which coordinate feed flowrates, will require information as to the impact on product quality and operational efficiency of the range of flowrates on individual process equipment. One of the advantages of continuous processing is the potential for operating plants to serve clinical development by shortening plant operation. Could the same be used in early stage process development? How does this scale match process development goals which, apart from producing material to demonstrate feasibility of the process, have broader goals such as generating envelopes of performance and experimental data for process understanding? This presentation will initiate discussion on early stage bioprocess development needs when facilities are running integrated continuous processes and envisage how the process of technology transfer from development scale to operating scale might look. We will provide insights into the challenges encountered in designing scale-down mimics of continuous unit operations such as tangential flow filtration or TFF [2] and will illustrate ultra scale-down concepts [3] which could be used to understand unit operations within a continuous platform. TFF is a key unit operation that has been cited as having potential for upstream cell separation or clarification. In a previous work [2], we successfully demonstrated a microscale TFF platform which mimicked a typical bench-scale TFF, Pellicon 2TM (Merck Millipore) based on operating conditions. We obtained similar fluxes, transmissions of antibody fragments, total protein and DNA (unpublished). This was achieved with membrane area that is smaller by 100-fold and reduced feed material by at least 10-fold. We identified that fluid transfer is a key limitation in the reduction of feed since pump requirements for continuous flow dictate the minimum volume of material needed to run the equipment efficiently. Without compatible fluid transfer technologies, material requirement for scale-down, continuous equipment will still be in the litre-scale per experiment. For investigative studies, important to identify key process parameters and quality attributes, these amounts would be prohibitive and would require more resources and time. This highlights the need to re-consider the typical use of geometrical scale-down models to evaluate continuous unit operations and requires more thought on early stage development. Otherwise, we may only be moving the cost and risk of biomanufacturing from industrial scale to the bench scale of process development. The USD approach endeavors to understand the complex engineering environment within an individual unit operation by identifying key engineering parameters and determining the critical flow regime. This insight is then developed into USD technologies and techniques to mimic larger scale operation. The approach suits the requirements during the early stages of product development when the amount of material is scarce and information about the product or process is limited. First applied to continuous centrifugation with the USD centrifugation technique, the USD concept has been extended for other unit operations. The USD techniques were powerful in revealing process interactions. They facilitate Quality by Design and help define process control strategy by determining and quantifying critical processing parameters which control the critical process attributes. References: (1) Konstantinov KB, Cooney CL. White Paper on Continuous Bioprocessing. May 20–21, 2014 Continuous Manufacturing Symposium. Journal of Pharmaceutical Sciences. 2015;104(3):813-20; (2) Rayat ACME, Lye GJ, Micheletti M. A novel microscale crossflow device for the rapid evaluation of microfiltration processes. Journal of Membrane Science. 2014;452(0):284-93; (3) Rayat ACME, Chatel A, Hoare M, Lye GJ. Ultra scale-down approaches to enhance the creation of bioprocesses at scale: impacts of process shear stress and early recovery stages. Current Opinion in Chemical Engineering. 2016;14:150-7

Development of a miniature bioreactor model to study the impact of pH and DOT fluctuations on CHO cell culture performance as a tool to understanding heterogeneity effects at large-scale

Understanding the impact of spatial heterogeneities that are known to occur in large-scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0 - 7.5 and DOT 10 - 30%, respectively, for durations of 15, 30 and 60 minutes. Fluctuations in pH had a minimal impact on growth, productivity and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5 however another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale-up. This experimental data can be combined with computational modelling approaches to predict overall cell culture performance in large-scale bioreactors. This article is protected by copyright. All rights reserved

Fed-Batch E. coli cultures in a shaken, single-use 24-well miniature bioreactor

At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes. Miniature bioreactors are becoming widely used in the biopharmaceutical industry as a tool for high throughput strain evaluation and fermentation process development. However, there are relatively few examples of miniature bioreactors capable of fed-batch operation and of supporting the high oxygen demand. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. In this work a shaken, single-use 24-well miniature bioreactor (Pall, Micro 24 MicroReactor System) has been characterised in terms of volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) to assess the feasibility of high cell density microbial cultures. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. To enable fed-batch cultivation in the miniature bioreactor system a bespoke feeding system for direct, continuous feed delivery has been developed that works at feed flow rates of 20μL h-1 and above. This feeding system allows for 24 fed-batch cultures to be run in parallel. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume; the oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency and gas flow rate over the range 3-101h-1. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.8-15.3s. It has been demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The bespoke feed delivery system was used to perform fed-batch cultures of an industrial E. coli strain producing an antibody fragment under operating conditions defined from the engineering characterisation studies. Fermentations were performed on a semi-complex medium containing glycerol with direct feeding of a glycerol solution initiated around 15 hours. It was found that direct feeding enhances biomass production by 30-40% and product expression by 45-65% in comparison to non-fed cultures. The feeding system developed in this work allows for industrially relevant microbial processes to be implemented at the microscale

Automated liquid-handling operations for robust, resilient, and efficient bio-based laboratory practices

Increase in the adoption of liquid handling devices (LHD) can facilitate experimental activities. Initially adopted by businesses and industry-based laboratories, the practice has also moved to academic environments, where a wide range of non-standard/non-typical experiments can be performed. Current protocols or laboratory analyses require researchers to transfer liquids for the purpose of dilution, mixing, or inoculation, among other operations. LHD can render laboratories more efficient by performing more experiments per unit of time, by making operations robust and resilient against external factors and unforeseen events such as the COVID-19 pandemic, and by remote operation. The present work reviews literature that reported the adoption and utilisation of LHD available in the market and presents examples of their practical use. Applications demonstrate the critical role of automation in research development and its ability to reduce human intervention in the experimental workflow. Ultimately, this work will provide guidance to academic researchers to determine which LHD can fulfil their needs and how to exploit their use in both conventional and non-conventional applications. Furthermore, the breadth of applications and the scarcity of academic institutions involved in research and development that utilise these devices highlights an important area of opportunity for shift in technology to maximize research outcomes

Microfluidic chromatography for early stage evaluation of biopharmaceutical binding and separation conditions

Optimization of separation conditions for biopharmaceuticals requires evaluation of a large number of process variables. To miniaturize this evaluation a microfluidic column (1.5 mu L volume and 1cm height) was fabricated and packed with a typical process scale resin. The device was assessed by comparison to a protein separation at conventional laboratory scale. This was based upon measurement of the quality of packing and generation of breakthrough and elution curves. Dynamic binding capacities from the microfluidic column compared well with the laboratory scale. Microfluidic scale gradient elution separations also equated to the laboratory column three orders of magnitude larger in scale

Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid

Large amounts of pectin-rich biomass are generated worldwide yearly, which can be hydrolysed by pectinases to obtain bio-based chemical building blocks such as D-galacturonic acid (GalA). The aim of this work was to investigate thermophilic pectinases and explore their synergistic application in the bioconversion of pectic substrates into GalA. Two exo-polygalacturonases (exo-PGs) from Thermotoga maritima (TMA01) and Bacillus licheniformis (BLI04) and two pectin methylesterases (PMEs) from Bacillus licheniformis (BLI09) and Streptomyces ambofaciens (SAM10) were cloned and expressed in Escherichia coli BL21 (DE3), purified and fully characterised. These pectinases exhibited optimum activity at temperatures above 50 °C and good stability at high temperature (40-90 °C) for up to 24 h. Exo-PGs preferred non-methylated substrates, suggesting that previous pectin demethylation by PMEs was necessary to achieve an efficient pectin monomerisation into GalA. Synergistic activity between PMEs and exo-PGs was tested using pectin from apple, citrus and sugar beet. GalA was obtained from apple and citrus pectin in a concentration of up to 2.5 mM after 4 h reaction at 50 °C, through the combined action of BLI09 PME with either TMA01 or BLI04 exo-PGs. Overall, this work contributes to expand the knowledge of pectinases from thermophiles and provides further insights into their application in the initial valorisation of sustainable pectin-rich biomass feedstocks

Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation

Background: The production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level. The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity. Results: Citramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%. Conclusions: This study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial ‘toolkits’ to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry

Potential of sugar beet vinasse as a feedstock for biocatalyst production within an integrated biorefinery context

BACKGROUNDThis work explores the feasibility of vinasse as an inexpensive feedstock for industrial biocatalyst production within the context of an integrated sugar beet biorefinery. As an exemplar, production of CV2025 ω‐Transaminase (ω‐TAm) in Escherichia coli BL21 was studied.RESULTSCharacterisation of vinasse showed that it comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 cell growth and CV2025 ω‐TAm production were feasible in cultures using 17% to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The d‐galactose present in vinasse facilitated auto‐induction of the pQR801 plasmid enabling CV2025 ω‐TAm expression without addition of expensive Isopropyl‐β‐d‐thiogalactopyranoside (IPTG). Assessment of different vinasse pre‐processing options confirmed simple dilution of the vinasse was sufficient to reduce the concentration of polyphenols to below inhibitory levels. Optimisation experiments, carried out using a controlled, 24‐well microbioreactor platform, showed supplementation of diluted vinasse medium with 10 g L−1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3‐fold in specific growth rate, maximum biomass concentration, CV2025 ω‐TAm volumetric and specific activity, respectively. Investigation into the metabolic preferences of E. coli BL21 when grown in vinasse showed a preference for D‐mannitol utilisation before simultaneous metabolism of glycerol, d‐xylitol, d‐dulcitol and acetate. Scale‐up of optimised conditions for batch CV2025 ω‐TAm production to a 7.5 L stirred tank reactor (STR) was demonstrated based on matched volumetric mass transfer coefficient (kLa). The results showed good comparability with respect to cell growth, substrate consumption and CV2025 ω‐TAm production representing over a 700‐fold volumetric scale translation. Further enhancements in CV2025 ω‐TAm production were possible in the STR when operated at higher kLa values.CONCLUSIONThis work describes the promising application of vinasse for production of microbial enzymes and insights into carbon source utilisation in complex feedstocks. Exploitation of vinasse as a fermentation feedstock could be further extended to other processes involving different microorganisms and target enzymes

Continuous enzymatic hydrolysis of sugar beet pectin and l-arabinose recovery within an integrated biorefinery

Sugar beet pulp (SBP) fractionated by steam explosion, released sugar beet pectin (SB-pectin) which was selectively hydrolysed using a novel α-l-arabinofuranosidase (AF), yielding monomeric l-arabinose (Ara) and a galacturonic acid rich backbone (GABB). AF was immobilised on an epoxy-functionalised resin with 70% overall immobilisation yield. Pretreatment of SB-pectin, to remove coloured compounds, improved the stability of the immobilised AF, allowing its reutilisation for up to 10 reaction cycles in a stirred tank reactor. Continuous hydrolysis of SB-pectin was subsequently performed using a packed bed reactor (PBR) with immobilised AF. Reactor performance was evaluated using a Design of Experiment approach. Pretreated SB-pectin hydrolysis was run for 7 consecutive days maintaining 73% of PBR performance. Continuous separation of Ara from GABB was achieved by tangential flow ultrafiltration with 92% Ara recovery. These results demonstrate the feasibility of establishing a continuous bioprocess to obtain Ara from the inexpensive SBP biomass