An ever increasing number of the new drugs currently under development are based upon proteins rather than traditional small molecules (e.g. antibiotics). These protein drugs are produced for the treatment of diseases such as cancer by bacterial, yeast or mammalian cells kept in culture under defined conditions. Many of the new antibody based drugs are produced from in vitro cultured mammalian cells in order to produce correctly folded and assembled and post-translationally modified products, these processes being essential to the bioactivity of these drug molecules. However, one problem with this approach is that the cells used to make such proteins secrete not only the target protein into the medium in which the cells grow, but other proteins from the cell as well, called host cell proteins (HCPs), and product impurities (incorrectly processed products e.g. half-antibodies), whilst product aggregates can also accumulate in the medium. Further, cell breakage during fermentation or downstream handling (e.g. centrifugation) can result in the release of intracellular protein material and product aggregates. To complicate things further, what these HCPs are (cellular the contaminants) and how they change throughout cell fermentation and with target products is not known. What this means is that the target drug must be purified from the rest of the material in the medium and other product related contaminants before it is deemed safe for use and this is referred to as downstream bioprocessing. During purification it is not usually known what HCPs or product impurities are removed by any given step and this makes the development of new or novel approaches particularly challenging. Almost all approaches to date also bind and elute the target protein, and there is scope for investigating disposable technology that binds the contaminants. Thus, whilst improvements in product yield are economically beneficial, this does not directly correlate with downstream processing and there is little economy of scale. This is largely due to the reliance of downstream bioprocessing approaches on multiple chromatography steps that are often complex and low yielding and it is the total mass of the product that determines the amount of chromatography resin required and therefore cost. Downstream bioprocessing is now a major part (>40%) of the total cost of manufacturing such drugs and as such improvements in this area would be of major benefit to both manufacturers and the NHS. This project will begin to address these areas and investigate the development of an integrated platform (upstream and downstream) for the production of recombinant protein biopharmaceuticals using disposable processing technology. Specifically, we will test the hypothesis that identification/characterization of the major host cell protein contaminants (HCPs) and product impurities in CHO cell culture and their interaction with the downstream process will allow the development of alternative knowledge-based disposable purification strategies and the rational engineering of host cell lines to limit the levels of such problematic HCPs and product contaminants. To achieve this we will use state-of-the-art proteomic technology at Kent and standard purification procedures developed by Pall to determine the fate and relationship of product, product impurities, and HCPs throughout the downstream processing of model recombinant proteins expressed in mammalian cells (CHO expression systems). Typically, we will use traditional 2D-PAGE based and non-gel based LC-MS based approaches at Kent to monitor the protein profile throughout a standard Pall purification process of a monoclonal antibody. We will then compare this to alternative downstream processes using Pall based disposable technology. We expect that these approaches will identify both the cell HCP contaminants and identify the recombinant protein degradation products and the heterogeneity of the recombinant material.

Live viral vaccines and therapeutics are growing in popularity due to their high specific potency yet their manufacture remains hampered by poor recoveries of viral infectivity following concentration and purification. In particular, practitioners highlight two such difficulties; the poor recovery of infectious virus following sterile filtration and the search for high yielding purification techniques. These related problems derive primarily from viral inactivation by fluid shear and interfacial phenomenon in the membrane modules and chromatography equipment commonly used by industry. We seek to understand the mechanisms of viral inactivation in order to design new processing systems that provide higher recoveries and are simple to use. Experiments will be conducted to characterise the key morphological and functional characteristics of representative enveloped and non-enveloped viruses (using Ad5 and MoMULV) following exposure to well characterised fluid shear typical of of that encountered during sterile filtration and chromatography. Measurement of viral infectivity will be made (TCID50) and electron microscopy, immunogold labelling and real time PCR will be used to assess the influence of shear on the external viral proteins that mediate infectivity. All these analytical techniques are available in the academic partner's laboratory where the general approach has been previously shown valuable for the study of viral inactivation during lyophilisation. The effect of stabilising excipients and rheology modifying agents will be examined . CFD data on shear distributions in commercial filter housings will be obtained to guide these studies. Sterile filtration yields will be assessed for prototype membranes with a range of porosities, morphologies and surface properties in order to assess the influenec of membrane characteristics upon viral inactivation. The distribution of representative nanoparticles on membranes will be studied using fluorescent labelled HSA particles and confocal imaging. As a result, housing designs will be modified to reduce any maldistribution of viruses. Prototype filter assemblies will be similarly probed with fluorescent immuno-labelled viruses and confocal microscopy. From these studies improved sterile filtration equipment and procedures will result. Complete prototype disposable virus manufacturing systems will be fabricated using collections of commercially available units. Typically, cell culture in disposable bioreactors, including Wave, will be linked by adsorptive and size based membrane processing cartridges and the manufacturing performances of these systems will be characterised for the test viruses. The academic partner is familiar with such approaches, having previously adopted similar approaches for the manufacture of antibody based snake antivenoms in a BBSRC sponsored project. The industrial partner is a market leader in filtration and separations and has relevant process separation technologies for exploitation in this area. Limitations in processing capability will be identified using existing systems and new systems designed to provide improved performance. We anticipate that radical improvements will emerge from the re-designed cartridge housings that result from data obtained on the influence of fluid shear on infectivity. We expect too that the understanding of interfacial phenomena together with new materials that are emerging from the laboratories of the industrial partner will enable significantly higher viral recoveries to be achieved.

De novo design and synthesis of affinity ligands mimicking natural biological recognition allows the purification of biopharmaceutical proteins, the resolution of isoforms and the extraction of low abundance proteins from the human proteome. The availability of crystallographic structures of proteins and complexes, together with refined computer-based molecular modelling techniques has lead to the concept of 'intelligent' design of chemically characterised, highly selective and stable affinity ligands for target proteins. Synthetic affinity ligands circumvent the drawbacks of natural IgG-binding ligands, such as resistance to chemical and biological degradation, and offer ease and low cost of production and in situ sterilization. In our previous work, highly selective adsorbents for biopharmaceutical proteins have been developed based on combinatorial libraries using a triazine scaffold. We propose now to concentrate on developing new methods for the purification of engineered antibodies, since it is predicted that by 2008, engineered antibodies will account for >30% of the total revenue in the biotechnology market. This has motivated us to design specific affinity adsorbents for the isolation of whole (IgG), monovalent (Fab, scFv) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) for the industrial-scale downstream purification of biomedical and research immunopharmaceuticals. We have recently developed a novel approach to protein fractionation which exploits peptoido-mimetic chemistry based on the 4-component Ugi-Passerini reaction. This multi-component reaction reacts an oxo-component, an aldehyde or ketone, generally immobilised to the solid phase, a primary or secondary amine, an isonitrile and a carboxylic acid in a 'one-pot' reactor to yield a single di-amide scaffold product. Multi-component reactions allow for substantial chemical diversity by incorporating 3, 4 or more reactants, each of which can be varied systematically to produce a variety of subtle changes to the final ligand structure. A particular advantage of the Ugi-Passerini chemistry for affinity ligand design is that this scaffold mimics the native dipeptide bond fairly precisely, with the interatomic distances between the O1-N-O2 in the native dipeptide being divergent from the Ugi scaffold by <1Å in a triangulated pharmacophore diagram. Both the carboxylate and amine substituents are directed away from the scaffold and therefore present an exploitable binding site for target interaction. The current list of commercially available reaction components from the Available Chemicals Directory (ACD) lists of amines, aldehydes, isonitriles and carboxylic acids, gives a potential combinatorial library of 3x1014 elements. We propose to construct limited (~100-200 member) solid-phase libraries of affinity ligands based on these 4-component reactions aimed at creating peptoido-mimetic ligands for binding immunoglobulins via the Fc (Protein A/G), Fab (Protein L) and glycomoiety, differentiating the various classes and sub-classes, binding various immunoglobulin fragments (scFv) and selectively binding immunoglobulins from several sources. We will use beaded Sepharose CL-6B and HyperCel as the aldehyde-substituted component and vary the other three components in an m x n array to generate a library of 'di-amide' type ligands covalently bonded to the matrix support. The binding behaviour of the target proteins will be confirmed by ELISA, small-scale (50microl) liquid chromatography and MS/MS. Those ligands exhibiting favourable IgG-binding characteristics will be re-synthesised using a larger scale suitable for further chromatographic evaluation. An iterative process of chemical synthesis, followed by biological evaluation, and complementation by molecular modelling, will lead to ligands displaying the desired level of specificity for whole and fragmented IgG whilst exhibiting negligible levels of host cell protein binding.

Recombinant protein and antibody biopharmaceuticals produced predominantly by mammalian cells in culture are a major class of therapeutic drugs, valued at $35bn pa. Transient expression technologies potentially enable the rapid manufacture from gene to protein of candidate recombinant therapeutic proteins by making product available in days rather than months. This supports the initial high-throughput in vitro studies to identify a therapeutic product and the production of larger quantities of recombinant protein for in vivo testing. There is now considerable industrial interest in this approach. Single-use, disposable biomanufacturing components for both upstream and downstream processes are accepted by industry as a viable route to reduce fixed and operating costs (e.g. capital equipment, cleaning) and to reduce space requirements. In broad terms, the objective of this proposal is to enable a student to develop a 'whole process vision' of integrated biomanufacturing using a combination of transient production and disposable bioprocessing. The project is targeted at the development of a rapid, low-cost manufacturing solution using a combination of innovations in upstream gene expression technology (Sheffield) and downstream bioseparation technology (Pall). Our model system will be the production of a monoclonal antibody in mammalian cell culture. We aim to design a novel disposable platform for rapid and low-cost protein production, a 'bioprocess on a bench'. Current technologies for transient production of recombinant proteins generally result in low volumetric product titres (1-20 mg L-1). To be of industrial use, it is necessary to significantly intensify transient production processes (100-500 mg L-1). Based on polyethylenimine-mediated transfection of CHO cells in chemically-defined media, DCJ's group has previously demonstrated that transient rMab production can be significantly improved by control of cell proliferation to maintain transcriptionally active rDNA in the host cell population for extended periods (Galbraith et al., 2006; Tait et al., 2004). Our model system will be based on commercially available CHO-S cells maintained in a chemically defined environment (Invitrogen) transfected with MAb-encoding plasmid vectors available in-house. To date, no study has attempted to improve transient productivity by simultaneous rational optimisation of numerous process parameters with discrete quantification and statistical analysis of their relative influence. The transient production process should be simple to implement, scalable, consume minimal rDNA, reproducible, cost-effective and GMP-compatible. For transient processes that consume plasmid DNA, we will include E.coli-based plasmid preparation as a sub-process in the overall process design and economic analysis. One challenge will be to integrate the upstream process stream with appropriately interfaced disposable processing components to generate a scaleable, robust 'plug and play' downstream process train capable of the necessary unit operations: particulate removal, product capture, concentration and purification. Pall have a large range of pre-packed disposable filtration products including direct or tangential flow, and chromatographic products both sorbents and columns and membrane chromatography devices. In addition Pall has a fully resourced R&D organisation available to this project to support new product developments in these areas. Bioseparation processes will be designed and optimised with respect to scalability (scale-up/down, out), cost-of-goods, speed/throughput, cumulative yield and product purity characteristics (e.g. alternatives to Protein A as an affinity sorbent, removal of gene delivery vehicle, host cell protein assays etc). An overall objective will be to balance process cost (costs-of-goods; labour time, materials etc) against product yield and purity to design a robust production platform.

The UK holds a leading position in the global life sciences scene. In this sector, biopharmaceuticals play a dominant role with almost £81bn in annual turnover (Life Sciences Competitiveness Indicators 2020, published: February 2021). Through the Life Sciences Vision 2021, the government is highlighting manufacturing innovation and ramp up as the UK's central aims. For the first time, Transition to Net Zero s brought at the centre of Life Sciences targets. For the UK to remain at the forefront of biopharmaceutical manufacturing, the Government is also encouraging digital innovation leading to time-/cost- efficient processes (Made Smarter, Review 2017). The crucial, positive health impact of (bio-) pharmaceutical processes may outweigh the environmental footprint of the sector that works with considerably lower volumes compared to other industries. Cumulatively, however, this remains to be an imminent challenge. Making those processes environmentally and economically sustainable is a complex task, involving conflicting objectives. For example, one would need to decide on the optimal number of separation cycles that meet both the target purity of the drug and create the least possible environmental footprint. Computer modelling tools can be of great help, lending themselves to the design and solution of multifactorial problems for the identification of the most suitable process setup and operating mode. In this respect, the research question this project aims to answer is: "How can we use computer modelling tools to embed environmental and economical sustainability in bioprocesses, while meeting the purity constraints?". In essence, the goal is to employ Engineering thinking and tools for the development of a systematic framework and software platform that will assist: (a) quantification of the impurity content on the downstream separation performance, (b) identification of a feasible and optimal design space, within which process performance is deemed satisfactory with respect to the tracked key performance indicators (KPIs) and (c) design of optimisation and control policies to ensure optimal operation. The novelty of the proposed work lies in two main aspects. Firstly, environmental sustainability KPIs, such as buffer and energy consumption will be considered for the first time systematically in the design of a bioprocess. Secondly, Engineering innovation will be deployed through the development of a computer modelling framework and software platform (i-PREDICT), harnessing the power of different modelling methodologies. In the junction of Engineering, Manufacturing, Digitalisation and Bioprocessing, i-PREDICT will enable bioprocess digitalisation and integration via continuous monitoring. This is one of the first computational attempts realising "Pharma 4.0" through the development and experimental validation of Industry 4.0-aligned frameworks for upstream in-process monitoring, optimisation and control. This work will create a roadmap towards the integration of product quality in the design of the bioprocess. Endorsing process intensification, this project proposes to consider upstream/downstream interplay through the quantification of the impact that impurity propagation in downstream. This novel concept will allow the design of variability-robust separation processes, enabling seamless unit integration and downstream scale-up. The digital and mathematical tools developed here will be validated experimentally, closing the loop from in silico to in vitro. This highly ambitious, multi-disciplinary project will create a step change towards a revolutionary research area of integrated design, optimisation and control in (bio-) pharmaceutical processes.