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 We obtained similar fluxes, transmissions of antibody fragments, total protein and DNA (unpublished).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

The role of membrane chemistry in Lentiviral vector clarification recovery for cell and gene therapies

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A scale-down model to investigate cell retention for continuous monoclonal antibody manufacture

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Production of indigo by recombinant bacteria

Indigo is an economically important dye, especially for the textile industry and the dyeing of denim fabrics for jeans and garments. Around 80,000 tonnes of indigo are chemically produced each year with the use of non-renewable petrochemicals and the use and generation of toxic compounds. As many microorganisms and their enzymes are able to synthesise indigo after the expression of specific oxygenases and hydroxylases, microbial fermentation could offer a more sustainable and environmentally friendly manufacturing platform. Although multiple small-scale studies have been performed, several existing research gaps still hinder the effective translation of these biochemical approaches. No article has evaluated the feasibility and relevance of the current understanding and development of indigo biocatalysis for real-life industrial applications. There is no record of either established or practically tested large-scale bioprocess for the biosynthesis of indigo. To address this, upstream and downstream processing considerations were carried out for indigo biosynthesis. 5 classes of potential biocatalysts were identified, and 2 possible bioprocess flowsheets were designed that facilitate generating either a pre-reduced dye solution or a dry powder product. Furthermore, considering the publicly available data on the development of relevant technology and common bioprocess facilities, possible platform and process values were estimated, including titre, DSP yield, potential plant capacities, fermenter size and batch schedule. This allowed us to project the realistic annual output of a potential indigo biosynthesis platform as 540 tonnes. This was interpreted as an industrially relevant quantity, sufficient to provide an annual dye supply to a single industrial-size denim dyeing plant. The conducted sensitivity analysis showed that this anticipated output is most sensitive to changes in the reaction titer, which can bring a 27.8% increase or a 94.4% drop. Thus, although such a biological platform would require careful consideration, fine-tuning and optimization before real-life implementation, the recombinant indigo biosynthesis was found as already attractive for business exploitation for both, luxury segment customers and mass-producers of denim garments

Insights into product and process related challenges of lentiviral vector bioprocessing

Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale-down technologies. The impact of shear during membrane filtration in a tangential flow filtration-mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems

A quality- by- design approach for the implementation of a manufacturing license change using a qualified scale- down process model

Influenza vaccines are required to be re formulated every year to account for antigenic drift with recommendations coordinated by the World Health Organisation. This results in a short development cycle of only six months to be able to choose and characterise a suitable reassortant of a recommended strain and proceed to commercial manufacturing of the influenza antigen and vaccine product. Due to the ongoing healthcare crisis brought about the Covid-19 pandemic, the demand for influenza vaccines has increased rapidly requiring vaccine manufacturers to be able to meet this demand and be the first to market in the season. This has necessitated the implementation of novel approaches to increase antigen and vaccine product yield in a rapid and yet robust process development. One of the potential yield improvements was the introduction of an optimised quantity of Hydrocortisone solution in the egg-based platform process at the inoculum stage for the Influenza A strains, which was already introduced and in use for Influenza B strains. In this presentation, we demonstrate the implementation of a scale-down modelling to support process changes and their subsequent regulatory approval. To facilitate the implementation of this change to the manufacturing license with various regulatory bodies, we devised a protocol to produce representative antigen batches at scale-down (i.e., 1% scale from a qualified area to the commercial batches) at higher and lower Hydrocortisone input on a variety of Influenza A strains. A total of 11 batches executed with various hydrocortisone inputs and Influenza A seasonal strains showed that the resulting antigen met internal drug substance batch release specifications and showed yield increase of 9-24% across various seasonal Influenza A strains, which could potentially be higher at the commercial scale. Please click Download on the upper right corner to see the full abstract

From flask to large scale high cell density production of ω-transaminase using auto- induction media

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Integrated ultra scale-down and multivariate analysis of flocculation and centrifugation for enhanced primary recovery

A linked ultra scale-down (USD) flocculation and centrifugation system facilitates rapid bioprocess development and evaluation. These techniques allow studies to be undertaken at high throughput using as little as 50 mL of feed material. They enable the investigation of multiple conditions, thus generating a relatively large amount of data. This study establishes USD flocculation and centrifugation and joins a sequential multivariate data analysis (MVDA) to evaluate the multiparameter effects on primary recovery performance. MVDA techniques such as Principal Component Analysis (PCA) and Partial Least Squares (PLS) were used to handle the complex data sets and investigate the relationships between process parameters and responses. A strategy to assess floc characteristics using PCA was proposed to eliminate the visual inspection, and aid in the analysis, of particle size distribution (PSD) datasets. The PSD of non-sheared and sheared flocs provide good indicators of floc centrifuge performance. The findings show that this USD system can be used to forecast pilot scale performance. The sequential analysis demonstrated that the produced flocs are shear-sensitive in which feed preparation and process shear significantly impact the centrifugation of those flocs. Strong flocs may not necessarily result from high Camp number values (≥ 105) where optimisation of the flocculation chemical parameters is required. The novel integration of the USD systems with MVDA is a powerful platform to optimise and expand process know-how

Microscale bioprocessing platform for the evaluation of membrane filtration processes for primary recovery

An automated microscale bioprocessing platform for membrane filtration processes was established to identify key process issues early and aid the rapid design of robust and scaleable filtration processes. To demonstrate the utility of this platform, it was used to investigate the impact of upstream operations on microfiltration performance. The primary recovery of humanised antibody Fab’ fragments from Escherichia coli (supplied courtesy of UCB Celltech) were used as a case study to evaluate the microfiltration methodologies and devices created in this work. Initially, the methodology associated with the microscale dead-end filtration device previously created and investigated by Jackson et al. (2006) has been improved by reducing the required volume by 50% (~500 \mu L). This improved method demonstrated reproducibility and sensitivity to changes in feed preparation. The method was then applied in the study of the influence of various cell disruption operations on subsequent solid-liquid separation and hence, Fab’ product recovery. Results showed that the heat extracted cells showed better dead-end microfiltration performance in terms of permeate flux and specific cake resistance. In contrast, the cell suspensions prepared by homogenisation and sonication showed more efficient product release but with lower product purity and poorer microfiltration performance. Having established the various microscale methods, the linked sequence was automated on the deck of the Tecan™ robotic platform and used to illustrate how different conditions during thermo-chemical extraction impacted on the optimal performance of the linked unit operations of product release by extraction and subsequent recovery by microfiltration. The microscale approach was then extended for crossflow operations. A microscale crossflow filtration device was designed to enable integration also within the Tecan™ platform for automated processing. The device has an effective membrane area of 0.001 m2, which is a hundred-fold smaller than the larger scale Pellicon-2™ membrane module used for scale translation studies, and has two independent membrane channels for parallel analysis. The device was first characterised by determining the normalised water permeability (NWP) of a Poly(vinylidene fluoride) membrane and compared this with the NWP of the membrane by dead-end filtration. NWP is an inherent membrane property and as expected, the NWP values derived from crossflow filtration experiments match the values derived from dead-end filtration to within 90%. For scale translation studies, two types of feeds were used: a model feed, which is resuspended active dry yeast and Bovine Serum Albumen in phosphate buffer, and the antibody fragment expressing E. coli strain. Results showed, that at matched optimal shear rates and transmembrane pressure, the percentage differences between microscale and large scale values were up to ± 25% for the permeate flux, ± 10% for Fab’ and total protein yields. These scale-up predictions were achieved with a ten-fold reduction in feed material requirement for crossflow operation. Overall, the results illustrate the power of microscale techniques to identify and enable the understanding of key process performance attributes in a bioprocess sequence. The broader implications derived from using these microscale membrane devices, further applications and recommendations for future research are also discussed

An ultra-scale-down method to predict diafiltration performance during formulation of concentrated mAb solutions

Formulation of monoclonal antibody (mAb) solutions using membrane filtration processing is a critical unit operation in the preparation of antibody therapies. A key constraint in formulation process development, particularly in the early stages of development and when using high protein concentration solutions, is the availability of material for experimental studies. Ultra-scale down (USD) technologies use a combination of critical flow regime analysis, bioprocess modelling and experimentation at the milliliter scale to enable a more effective process development approach significantly reducing process material, cost and time requirements (Rayat et al, 2016). The ability to predict the performance of large-scale (LS) operations, e.g. flux profile characteristics and changes in protein structure, will help maximize the value of eventual high cost pilot-scale runs during process development. In this study a USD membrane device, comprising a sheared cell unit with a rotating disc and with an effective membrane area of 0.00021 m2 developed at University College London, is used to predict the performance of a LS cross-flow membrane cassette of area 0.11 m2. The USD set up was designed to mimic the LS in terms of processing volumes, membrane area and process times. Computational Fluid Dynamics (CFD) is implemented to characterize average shear rates as a function of suspension viscosities and disc speed of the USD membrane device. A series of trials at USD scale established the effect of average shear rate on flux and the rate of flux decline during a diafiltration operation reaching 7 diafiltration volumes. A series of LS runs were carried out at different cross flow rates covering a similar range of average shear rates as the USD trials. Good correlation was obtained between USD and LS performance using constant average shear rate over the membrane surface as the basis for scale translation between the two scales of operation. The predicted effect of change in shear rate on flux in USD matched that found in LS. This scale correlation on performance was additionally verified by studying the effect of type and concentration of mAb. The comparable process performance was achieved at USD with 520-fold reduction in effective membrane area, required process material and diafiltration buffer for the trial. Future studies will include membrane concentration operations and evaluating sensitivity to stress-related effects and the impact of operation at higher protein concentrations. Rayat, A.CME; Chatel, A; Hoare, M; Lye, G.J (2016). 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 14:150-15