Rehabilitation of old water supply pipes

Rehabilitation of old water supply pipe

Rapid vaccine development using a micro-scale platform

Vaccine research and development is becoming increasingly important because of the potential to create a blockbuster drug, such as Prevnar. However, the development pipeline continues to be a limiting factor in commercialising a vaccine. In this thesis a micro-scale platform is created to mimic the key features of a unit operation so that it is possible to calculate the impact of a commercial manufacturing process using this scaled down platform. Two model vaccines were applied to the micro-scale platform, a new Meningitis serogroup B vaccine based on the outer membrane vesicle proteins of Neisseria lactamica and the licensed UK Anthrax vaccine. To create the platform, cultures of Neisseria lactamica in microwells have been combined with statistical techniques such as Design of Experiments to increase biomass production by four fold and antigen yields by 165%. Microwell experiments were coupled with SELDI-TOF mass spectroscopy to enable a detailed insight into the changing vaccine composition with culture conditions. Microwell results here were scaled up to 2, 8 and 50 litre fermentations using dimensionless analysis based on the oxygen mass transfer co-efficient, kLa. The effects of pilot scale downstream processing were investigated using ultra scale down tools and models. It was possible to characterise product losses and the robustness of the process stream by conducting shear experiments. Furthermore, final product filter sterilisation was investigated using a microwell platform coupled with statistical analysis, particle sizing and DLVO theory. Through these studies it was possible to minimise aggregation and increase antigen transmission through the membrane from 35% to 78%. The platform was applied to cultures of Bacillus anthracis Sterne 34F2, the Anthrax vaccine strain. Microwells were used to mimic Thompson bottle cultures and ascertain the main factors which effect B. anthracis growth and antigen production. The cell density dependent signalling mechanism, known as quorum sensing was found to control growth and antigen production in B. anthracis and that a protein below 5kDa may be involved in the quorum sensing mechanism along with the auto inducer molecule, AI-2. Finally, transfer of B. anthracis vaccine production from static culture to a homogenous stirrer tank culture environment was investigated using a miniature bioreactor. It found that transfer was possible and that doing so reduced the culture time from 28 hours to just 14 hours, increasing production of PA and LF vaccine antigens by 25% and 78% respectively. Aeration of the culture showed that biomass production could be improved upon, but it had a detrimental effect on antigen expression

Fully automated high-throughput process development for the novel purification of rotavirus vaccines

Downstream processing of biopharmaceuticals, such as immunoglobulins, recombinant proteins and protein-based vaccines, traditionally requires multiple purification steps that can introduce cost and time-related limitations. To avoid these, alternative purification strategies are sought involving novel and efficient processes. Mixed-mode chromatography based unit operations can achieve these by making use of their multimodal functionality. This adheres to the goal of the Ultra-low cost Transferable Automated Platform for Vaccine Manufacture (ULTRA) project and its ultimate aim to deliver vaccines with a cost of goods less than 15 cents per dose. Herein, we show the application of mixed mode chromatography for the purification of a P2-VP8 (PATH) rotavirus antigen vaccine expressed in Pichia pastoris (MIT, MA, USA; J. Christopher Love). A high-throughput workflow was adopted for the development of the purification step which employed microscale chromatography with miniature columns. This was implemented on an automated liquid handling station (Tecan Evo 200). The performed scouting studies focused on combinations of different binding and elution conditions along with multiple resins and selectively accessing the ion exchange and hydrophobic integration mechanisms of the resins. Therefore, a wide range of conditions were assessed with walk-away automation. Conditions leading to high yields and purities were then scaled-up for verification purposes. This provided further evidence for the good scalability properties of the miniature chromatography column technique. The results from this study demonstrate that mixed mode chromatography can potentially lead to the establishment of a highly desirable, one-step purification of rotavirus vaccine upon further optimisation. Hence, downstream processing in rotavirus vaccine production can be de-bottlenecked with systematic process development activities leading to significantly improved whole process efficiencies. The utility of this method is that it has generated reproducible and scalable data with reduced sample requirements to just a few millilitres very early on in the development process. More broadly, this high-throughput methodology will enable the early purification screening of multiple vaccine candidates and enable their selection for further development based on ease of processing

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed-batch fermentation.

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)

Molecular engineering improves antigen quality and enables integrated manufacturing of a trivalent subunit vaccine candidate for rotavirus

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

Macroscopic modeling of bioreactors for recombinant protein producing Pichia pastoris in defined medium

© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC 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

Mechanism of Thimerosal-Induced Structural Destabilization of a Recombinant Rotavirus P[4] Protein Antigen Formulated as a Multi-Dose Vaccine

© 2020 The Authors 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