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 We obtained similar fluxes, transmissions of antibody fragments, total protein and DNA (unpublished).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

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

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