Bioprocessing 101: Cells to proteins, operations to processes, control to quality

This presentation will provide an initial introduction for delegates attending the pre-conference workshop on Basics of Biotechnology [1, 2]. It is designed primarily for those from physical science and traditional engineering backgrounds wishing to gain a rapid overview of biological products and methods for their manufacture. It will also be useful for life scientists wanting to gain insights into the design, monitoring and control of bioprocess unit operations and whole process sequences. This information will be presented in the context of the required product quality characteristics and their relationship to regulatory requirements. The first part of the presentation will focus on different types of protein-based products, e.g. therapeutic enzymes, vaccines and antibodies, their basic structure and analytical techniques for their characterization. It will then discuss the features of different expression systems, such as microbial and mammalian cells, their growth requirements and suitability for producing different types of protein product. Fermentation or cell culture is at the heart of any biomanufacturing processes. This part of the talk will give an overview of fermentation and cell culture process development focusing initially on growth media design and optimization. It will then discuss key features of traditional stirred bioreactors and how their operation impacts on cell growth and product formation. Bioreactor monitoring and control will also be addressed with particular emphasis on the types of probe conventionally used for monitoring culture conditions such as pH, temperature and dissolved gasses (O2 and CO2). The final part of the presentation will give an overview of the different stages of downstream processing from primary product recovery to purification. It will outline the key unit operations used at each stage (centrifugation, filtration, chromatography) and their role in removing key product and process related impurities and contaminants. It will also address the synthesis of typical downstream process sequences in relation to meeting economic and regulatory requirements such as cost and purity. This presentation is designed to lay the foundations for the following talks focused on the manufacture of antibody and cell therapy products. These will address the specific features and therapeutic applications of these key product classes and their industrial manufacture. These subsequent talks will also address the drivers and challenges associated with the introduction of single-use bioprocess technologies for their manufacture. [1] Ratledge, C. and Kristiansen, B. (2006) Basic Biotechnology, 3rd Edition, Cambridge University Press. [2] Doran, P.M. (2012) Bioprocess Engineering Principles, 2nd Edition, Academic Press

Microbial applications of Single Use bioreactor systems

This work describes the ongoing assessment of single use bioreactors (SUBs) and their suitability for culturing microbial expression systems. Microbial expression systems are responsible for significant revenues as part of the biotechnology industry, in fact, their projected value for 2016 is in excess of $250 billion [1]. This promotes further interest in the area, with novel expression systems and pathways constantly under construction for the generation of bulk and high value chemicals. The advantages offered by single use bioreactor technology has lead to increased adoption in many areas of bioprocessing [2], and microbial fermentation is no different. Applications include simple fermentation, but additionally areas such as whole cell bioconversions, which can further increase the challenges already presented to single use systems. Arguably the greatest of these challenges is providing sufficient oxygen mass transfer [3], not only to support the culturing of microbial expression systems, but also for use as a substrate during processing with whole cell oxidative bioconversions. In order to facilitate the processing of microbial whole cell oxidative bioconversions in single use bioreactor technology, a whole cell P450 bioconversion was optimized at small scale using multifactorial statistical methods and characterized in a traditional bench top stirred tank bioreactor (STR). Characterisation of this elevated oxygen requirement, as a result of growth and the bioconversion forms the basis for understanding the demands that will be placed on SUBs. The process of characterizing the oxygen mass transfer capabilities of a range of SUB systems is also underway. This is being done using static and dynamic methods, but also by modeling the influence of a range of key process factors. This characterisation is being carried out in bag type systems, as well as in more rigid technologies, which more closely resemble traditional STRs such as the Ambr250

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

Klebsiella pneumoniae as cell factory for chemicals production

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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

Single-use bioreactor technologies for early stage development of microalgae cultivation processes

This work describes the engineering characterization and evaluation of two novel, single-use photobioreactor technologies for microalgal cultivation. There is currently considerable interest in microalgae as alternative expression systems for the production of value-added chemicals as well as therapeutic proteins and vaccines. In contrast to mammalian expression systems, however, bioreactor platforms to address early stage development challenges are not well established particularly for phototrophic and mixotrophic cultivation strategies. To support early stage cell line selection and process characterization a single-use, 24-well micro photo-bioreactor (mPBr) was established together with an illuminated and environmentally controlled shaker platform [1]. The same orbital shaker platform was also used for microalgae cultivation up to 10L scale, in single-use bags usually used for mammalian cell cultivation on rocked platforms [2]. Particularly for small and micro-companies this single-use photobioreactor (SUPBr) provides a route for rapid process optimization and materialization of novel products from microalgae. Both bioreactor technologies were characterized in terms of their fluid hydrodynamics, mixing and gas-liquid mass transfer. The influence of these factors as well as light path length and light intensity on growth and pigment formation in Chlorella sorokiniana was also studied. Successful scale translation between the mPBr and the SUPBr was demonstrated illustrating the complementarity of the two approaches to help reduce microalgal bioprocess development timelines

Mixing and fluid dynamics characteristics in single-use bioreactors for improved design and scalability

The pharmaceutical industry is at the forefront of the production of antibodies using mammalian cell-based cultures, with single-use technologies gaining prominence in the manufacturing process. Since the development of the first rocked bag bioreactor in 1997, other novel designs have been developed such as orbitally shaken, two and three dimensionally rocked, pneumatically driven, in addition to inflated cylindrical stirred bags. These have the potential to address new applications like expansion of adult stem cells for allogeneic therapies approaches by providing sufficient mixing while controlling maximum shear stress levels. Rigorous fluid dynamics studies are needed to understand the flow behaviour at different operating conditions, be able to determine meaningful dimensionless characteristics and establish robust scaling laws. A combination of different advanced analytical techniques were used to determine mixing and velocity characteristics and the validity of the approach is demonstrated by three case studies looking at different bioreactor flows relevant to bioprocessing. In this work, phase-resolved Particle Image Velocimetry (PIV) and high frequency visual fluid tracking were used to investigate the flow pattern and mixing characteristics of a geometrical mimic of a Sartorius 2L CultiBag at various rocking speeds and fill volumes. Fluid velocity and rate of dissipation of the turbulent kinetic energy were found to significantly vary with rocking speed. Under specific experimental conditions, wave formation was observed, which corresponded to high gas transfer rates from the headspace into the liquid phase. Higher rocking speeds caused the fluid to move proportionately out of phase with respect to the platform. Dimensional comparisons of fluid velocities with similar volume conventional bioreactors suggest that similar fluid dynamics characteristics can be achieved between rocked and stirred bioreactor configurations. Large scale orbitally shaken bioreactors employ the agitation principle of shaken flasks and microwell plates, providing a single-use upstream process thus facilitating scaling-up and simplifying regulatory approval. In this case study the mixing and flow dynamics in a cylindrical orbitally shaken bioreactor with conical bottoms of different heights was evaluated. The rationale for a conical bottom is to ease the suspension of cells or microcarriers for adherent cells applications. This study builds upon previous works of the research group (Weheliye et al 2013, Rodriguez et al. 2013, Rodriguez et .al. 2014, Ducci and Weheliye, 2014) for flat bottom reactors, where increases in Froude number were found to determine a mean flow transition and to increase the turbulence levels. PIV and Dual Indicator System for Mixing Time, DISMT, combined with advanced image processing were employed to assess the mixing performance in the bioreactor with a conical bottom and the findings offer a novel approach to design the next generation of products and improve scaling methodologies for cell therapy applications involving microcarriers’ suspensions. Thirdly, a thorough experimental study of the flow within the Millipore 3L CellReady stirred reactor was conducted combining PIV with a biological study into the impact of fluid dynamic characteristics on cell culture performance. PIV measurements conveyed a degree of fluid compartmentalisation resulting from the up- pumping impeller. Both impeller tip speed and fluid working volume had an impact upon the fluid velocities and spatial distribution of turbulence within the vessel. Cell cultures were conducted using the GS-CHO cell-line and a significant reduction in recombinant protein productivity was found at conditions corresponding to the highest Reynolds number tested in this work. 1. Rodriguez G, Pieralisi I, Anderlei T, Ducci A, Micheletti M. Appraisal of fluid flow in a shaken bioreactor with a conical bottom at different operating conditions. Chem Eng Res Des. 2016;108:186–197. 2. Odeleye AOO, Marsh DTJ, Osborne MD, Lye GJ, Micheletti M. On the fluid dynamics of a laboratory scale single-use stirred bioreactor. Chem. Eng. Sci. 2014;111:299–312