Continuous integrated biologics manufacturing

Biosimilars and patent expiry are forcing the biopharma industry to find new ways to maintain competitiveness by ensuring affordability, quality, and delivery performance. Despite great improvements in upstream processing (USP) efficiency, higher titres create downstream processing (DSP) bottlenecks and facility fit issues: Equipment reaches its physical and capacity limits thereby increasing processing time, material consumption and overall cost. Continuous processes have been proposed as a solution to many of these issues as they offer higher productivity while reducing cycle times, buffer/resin usage and required footprint. A consortium of UK based biopharmaceutical companies, suppliers and not for profit research organisations, funded by an Innovate UK grant, has been created and will investigate how such an integrated, continuous downstream process system can be realised. The system has been constructed and is currently operating at the Centre for Process Innovation at Darlington, UK. The project combines and condenses multiple DSP unit operations to function as one uninterrupted system with integrated analytics and overarching automated control. The aim is to create an operationally-efficient, multi-product platform which replicates the functionality of a larger plant processing 100 L feedstock per day (independent of product titre). The integrated unit will be tested on several biologic processes demonstrating the system’s potential to enable product changeover, increased facility flexibility and productivity. Significant focus will be given to process validation procedures and the use of low level control to achieve process stability (steady state) and maintain acceptable product quality. This work will lay the foundation for real-time release strategies and replace drug substance release testing. This presentation will provide an overview of the project and show recently-acquired data from the automated purification of industry-relevant monoclonal antibodies. In doing so, this will highlight the applicability and demonstrate the real-world potential for integrated continuous processing to advance the manufacturing of biopharmaceuticals

Developing a flexible automated continuous downstream processing system for research to clinical supply

Continuous manufacturing has gained a lot of attention over the last 10-15 years for numerous reasons such as the potential for higher efficiencies, reduced cost of goods, and improved product quality. However, the adoption of these technologies has been slow due to concerns over operating these processes in a GMP manufacturing environment. Some of these concerns relate to the operation of multiple continuous unit operations in an integrated process sequence. This presentation will highlight these concerns and show how these issues were addressed by developing an overarching automated and modular platform which can be easily adapted for processing most products. The developed automation platform is the result of a project funded by Innovate UK that brings together a number of biopharmaceutical companies including Allergan, AstraZeneca, Fujifilm Diosynth Biotechnologies and GSK to identify and address these issues. One objective of the project is to develop a flexible automated biologics downstream process consisting of multiple unit operations that can be rapidly reconfigured for manufacturing different products. To that end the process has been design with modularity in mind with each module having common inputs and outputs. The automation software has also been developed in a way that most typical downstream processes can be implemented in the system with little to no software updates. The ability to rapidly reconfigure the process has been demonstrated by using the system to produce two products with different process sequences. Another issue that inhibits the adoption of continuous technologies is the concern over simultaneously operating multiple unit operations. This presentation will detail how the automation software was developed to control both the key unit operations such as chromatography and filtration steps but also intermediate operations such as feed conditioning and viral inactivation steps. The automated system reduces the complexity of downstream processes, which can have in excess of eleven unit operations, to a single user-friendly interface. Implementing this control platform enables a single operator to control the entire process. This presentation will also detail how the automation strategy has been developed to enable a single operator to deal with start-up/shutdown, perturbations in the process and mid-process equipment turnover. It will highlight the challenges that have been faced when developing this system and how these have been overcome. The aim of this project was to improve efficiency by reducing processing time when compared to the current batch process and this was demonstrated by testing the system with two different products (a MAb and a MAb fusion protein). Furthermore, this presentation with show data from the production of these two products that demonstrates comparability between the continuous process and the original batch processes

Design and characterisation of food grade powders and inks for microstructure control using 3D printing

Additive Manufacturing techniques have been previously applied to food materials with direct consumption in mind, as opposed to creating structural ingredients as shown in this study. First, semi-crystalline cellulose was mechanically treated by ball milling to render an amorphous powder, which has been characterised. Requirements for the subsequent recrystallization of this powder with a view to structuring have been determined through the control of moisture and thermal energy. Food inks based on xanthan gum have been formulated to enable successful jetting with a FujiFilm Dimatix ink jet printer. The polymer inks were subsequently jetted onto the amorphous cellulose powder to observe powder-binder interactions. Material combinations and parameters were optimised to produce cohesive geometric structures. The results of this study are promising when looking towards using these materials in a binder jetting additive manufacturing technique using designer particles and inks to create structures for use in food products

Cycling comfort on asphalt pavement: Influence of the pavement-tyre interface on vibration

Attainment of cycling comfort on urban roads encourages travelers to use bicycles more often, which has social and environment benefits such as to reduce congestion, air pollution and carbon emissions. Cycling vibration is responsible for the cyclists’ perception of (dis)comfort. How asphalt pavement's surface characteristics relate to cycling comfort, however, remains undiscovered. In this study, the cycling vibration intensity on 46 sections of 24 urban roads was tested using a dynamic cycling comfort measure system while the cyclists’ perception of vibration was identified via questionnaires; the cycling comfort was then defined based on the cycling vibration. To record the accurate pavement-tyre interface under a stable environment, a total of 19 pavement sections were scanned using a 3D digital camera. These 3D models were then 3D printed, which are used to conduct the pressure film test using a self-developed pavement-tyre interface test system. Three ranges of pressure films were adopted to characterize the pavement-tyre interface via 9 parameters, namely contact area (A c ), unit bearing area (B u ), stress intensity (S i ), stress uniformity (S u ), kurtosis (S ku ), spacing (Sp a ), maximum peak spacing (Sp max ), radius ratio (R r ) and fractal dimension (F d ), in consideration of the area characteristics, pressure amplitude, peak spacing and shape of the interface. Finally, the significant interface parameters were identified, and the regression model between interface parameters and cycling comfort was established. Results show that the cycling vibration was described to be ‘very comfortable’ when the human exposure to vibration level (a wv ) was less than 1.78 m/s 2 ; ‘comfortable’ when the a wv was between 1.78 m/s 2 and 2.20 m/s 2 ; and ‘uncomfortable’ when the a wv was greater than 2.20 m/s 2 . The average stress on rear wheel-pavement interface is higher than that of the front wheel. B u-0.6 , Sp a-0.6 , and F d-0.6 are significant to cycling vibration. The 2LW pressure film is recommended for use to measure the bicycle pavement-tyre interface. The recommended interface characteristics are less than 7 mm 2 of the unit bearing area, 6 mm of average spacing and 2.38 of fractal dimension. Finally, dense asphalt mixture performs better in providing cycling comfort than the gap-graded asphalt mixture. Results of this study contribute to current knowledge on bike lane comfort and pavement design, the findings should be interested in cyclists, transport planners, and road authorities

Development of Endogenous and Synthetic CHO Promoter Expression Systems for Recombinant Protein Production

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell expression system for the industrial production of biotherapeutic recombinant proteins. Mammalian expression systems are a popular choice for the production of complex biopharmaceuticals due to their ability to correctly fold and assemble recombinant proteins and to carry out necessary human-like post-translational modifications. Traditionally, viral promoters are used to drive transgene expression in mammalian cells. This study reports on the investigation of endogenous CHO promoters as potential alternatives to the commonly used strong viral promoters such as SV40 and CMV. Although these promoters provide high recombinant gene expression, this is not always favourable as constitutive transgene over expression can ultimately lead to cellular stress. Furthermore, viral promoters have been reported to undergo silencing mechanisms over long culture periods, compromising product titers. Endogenous and synthetic CHO putative promoter sequences were investigated for their abilities to drive reporter and recombinant gene expression both in stable and transient systems. Initially a panel of ten putative promoters were identified from the literature, these being identified for this study as the associated gene or protein had been reported to have high transcript or protein amounts in CHO cells. The sequence 400 bp upstream of the transcript or translation start site of these genes was cloned into a promoterless eGFP reporter vector and their ability to drive transient gene expression examined. The ability of these putative promoters to drive eGFP expression was generally inferior to that of the viral SV40 promoter and always many-fold lower than that observed from the viral CMV with enhancer promoter. Regions 2 kb upstream from the transcript or translation site for each gene were then cloned into the promoterless eGFP reporter system to assess for promoter activity further upstream. For one of the 2 kb sequences, eGFP expression was enhanced above that observed from the equivalent 400 bp sequence and was greater than that from the SV40 promoter. A further nine target putative promoters were then identified from published RNAseq studies and sequences 500 bp upstream of the transcriptional start site of identified genes were cloned. Of the nine 500 bp sequences, three displayed promoter activity equivalent to or above that of SV40 and so regions 2 kb upstream of the transcriptional start site of these genes were investigated for further promoter activity. The use of the 2 kb segments resulted in an increase in stable but not transient eGFP expression from that observed from the 500 bp sequences alone. When placed directly downstream of the CMV enhancer two of the 2 kb sequences showed higher eGFP expression than when the CMV enhancer was not present. The CMV enhancer had no impact when upstream of the two other 2 kb sequences investigated showing this to be a sequence dependent effect. The 2 kb sequence which exhibited the strongest promoter activity of targets when driving transient eGFP expression, in addition to the two sequences with the CMV enhancer which showed enhanced transient eGFP expression, were investigated for their ability to drive IgG heavy and light chain, and hence IgG, stable expression in CHO cells compared to the CMV promoter with enhancer. Upon generation of pools and mini pools stably producing IgG under the control of the various promoters, the CHO endogenous 2 kb promoter outperformed the synthetic candidates, the CMV with enhancer and an industrially relevant control promoter. Indeed, the endogenous CHO promoter sequence was shown to achieve product titers of up to 3-fold greater than those from the commonly used CMV promoter with enhancer in CHO cells, demonstrating the potential for endogenous promoters to replace typically used viral promoters for recombinant gene expression. Further, colonies emerged faster after transfection and selection when using this promoter compared to the others investigated and a larger range of higher-expressing pools were available for investigation. In summary, this study has identified an endogenous CHO promoter sequence able to drive IgG expression beyond that of current widely used viral promoters and has generated additional promoters exhibiting a range of abilities to drive recombinant gene expression to varying amounts that could be applied to cell engineering approaches in the future

Methacrylate homo and copolymers with controlled architecture as adhesive viscosity modifiers in UV curable ink-jet inks

Reversible addition-fragmentation chain transfer (RAFT) controlled polymerisation was used to prepare a range of methacrylate polymers;- poly(methyl methacylate) (PMMA), poly(n-butyl methacrylate) (PnBMA), poly(hexyl methacrylate) (PHMA) and poly(methyl methacrylate)-co-poly(n-butyl methacrylate) (PMMA-co-PnBMA) with a range of molecular weights. The resulting polymers were characterised by 1H and 13C NMR spectroscopy and the molecular weights and distributions determined by gel permeation chromatography (GPC). The viscosity modification ability and the effects on adhesion and wettability were evaluated. Polymers were dissolved into a UV-curable ink formulation composed of a pthalocyanine blue pigment dispersion, monomers n-vinyl caprolactam, phenoxy ethylacrylate and photo-sensitive initiators;- 1-hydroxy cyclohexyl phenyl ketone, acyl phosphine oxide and benzophenone. An evaluation of the surface energies and polar and dispersive components of a range of thermoplastic printing media was undertaken using static contact angle analysis. Dynamic contact angle and adhesion testing was performed on typical printing media of high and low surface energy (dynes cm-1). The addition of the PMMA, PnBMA and PMMA-co-PnBMA copolymer enabled control over viscosity both by concentration and molecular weight. PHMA provided minimal control over viscosity at 3 wt% addition with varying molecular weight. Improved adhesion to particular grades of polypropylene was achieved with the addition of PnBMA, however a reduction in adhesion to poly(vinyl chloride) was also observed. The low molecular weight PMMA-co-PnBMA copolymer at 3 wt% achieved marginal improvement in adhesion to the printing grades PP without detrimental effects to adhesion of the PVC grades. The effect on polymer architecture therefore, appears to have a significant effect on the level of adhesion achieved under the test conditions outlined within the project. Although a significant correlation between molecular weight and viscosity was observed no significant difference was detected between adhesion and molecular weight. Measured surface free energy (ys) using static contact angle is an effective method of determining the true surface characteristics of printing substrates; however this study determined that a correlation to high and low surface energy and good and poor adhesion is not easily identifiable

The application and validation of high-throughput methods in Chinese hamster ovary cell line development

Chinese hamster ovary (CHO) cells are the main production platform for biotherapeutic proteins. In this thesis, two areas were identified and investigated with improving recombinant protein production from CHO cells and cell line development; transcriptomics and technologies for industrial cell line development (CLD). Transcriptomics wise, publicly available data were identified and analysed in order to identify 'common' transcriptomic signatures of cell growth or productivity in order to devise novel cell line engineering strategies. From the literature, 19 different transcriptomic datasets were aggregated that explored the differences between high productivity and fast growth phenotypes. Here, we proceeded to analyse the data in terms of the two simplest dimensions - the frequencies of genes appearing across these data sets and the concordance (the arithmetic mean of expression values) with regard to cell growth (µ) and productivity (Qp). By mapping out the contributing genes it was possible to construct a transcriptomic 'fingerprint' of a high-performing cell line. After identifying the most common and concordant genes, those genes that had a frequency of two or more were analysed using a pathway enrichment algorithm. From this it was identified that the cell cycle and lysosome pathways are significant targets for cell line engineering. To our knowledge, this effort is the first of its kind within CHO transcriptomics. CLD involves labour and resource intensive cloning out a genetically diverse pool of cells engineered to produce the protein of interest. We sought to analyse a new single cell analysis methodology (Berkley Lights Beacon, BLB) against an industrial ClonePix™ 2 CLD process adapted from FUJIFILM Diosynth Biotechnologies (FDB). We found that there were no statistically significant differences between cell groups generated from the BLB or ClonePix™ 2 processes. Using the Beacon® system, it was possible to predict 3 out of the top 5 producing clones for both Etanercept and Blosozumab. Within the standard ClonePix™ 2 CLD group of cell lines, predictions were most accurate from 24-well plate fed-batch and TubeSpin® batch culture ranks. Further, using the BLB, the time from recovery from transfection to cultures that were ambr® ready was reduced from 65 days to 42 days. Based on the findings of this research it is proposed that the Beacon® is an attractive and powerful new tool in industrial cell line development efforts. To the authors knowledge, this is the first in depth work validating a next-generation CLD process in such detail