CCE Award Lecture - Cell Culture and Social Engineering

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Deepening Knowledge on CHO cells metabolism using multiple tracer substrates

Optimization of culture conditions through trial-and-error has led to much of the past progress on animal cell culture. Today, a renewed account of the underlying biologies through a “systems” view is providing a deeper knowledge of cellular physiology and laying the basis to reengineer desirable states of further increased culture performance. In this work, we took a comprehensive view of cellular metabolism by extensively profiling extracellular and intracellular metabolomes after 13C‑label supplementation in parallel labelling cultures of [1,2-13C]Glucose, [U-13C,15N]Asn, [U-13C,15N]Ser and [1-13C]Pyruvate. This integrative approach was used to trace metabolic rearrangements in different scenarios of asparagine and serine availability in GS-CHO cells. The absence of asparagine in the medium caused growth arrest, and was associated with a dramatic increase in pyruvate uptake, a higher ratio of pyruvate carboxylation to dehydrogenation and an inability for de novo asparagine synthesis. The release of ammonia and amino acids such as aspartate, glutamate and alanine were deeply impacted. This confirms asparagine to be essential for GS‑CHO cells as the main source of intracellular nitrogen as well as having an important anaplerotic role in TCA cycle activity. In turn, serine limitation also negatively affected culture growth while triggering its de novo synthesis, confirmed by label incorporation coming from pyruvate, and reduced glycine and formate secretion congruent with its role as a precursor in the metabolism of one‑carbon units. The results obtained suggest that feeding schemes of asparagine or serine should be tightly tuned to minimize by-product formation while assuring biosynthetic needs. Ongoing is the contextualization of the data from the parallel labelling cultures into a metabolic network model for detailed mapping of cellular fluxomes in each scenario. Overall, this work contributes to unfold important insights into GS-CHO cells metabolism, and can be used as a basis for exploring bioprocess optimization strategies

Process intensification for production of a Peste des Petites Ruminants Virus (PPRV) vaccine

Peste des Petites Ruminants Virus (PPRV) is a highly contagious disease affecting small ruminants in Africa and Asian countries, with negative/significant economic impact. Aiming to eradicate the disease, a novel/scalable PPRV vaccine production process is clearly needed. Built upon work previously done at iBET, a new production process is herein proposed using Vero cells growing on microcarriers, serum-free medium (SFM) and stirred-tank bioreactors (STB). This includes a new method for cells detachment from microcarriers, and perfusion culture for reducing turnaround time. The PPRV vaccine production process was developed in 2L STB (BIOSTAT DCU-3, Sartorius) using Nigeria 75/1 strain. Engineering correlations (e.g. shear stress and Eddy size) were used to optimize culture conditions. Vero cells were adapted to grow in ProVeroTM-1 SFM (Sartorius). A new enzymatic and mechanical method for in situ cell detachment from microcarriers was designed. PBS washing, TrypLE Select and trypsin inhibitor concentrations, and stirring rates were the variables explored. Perfusion culture was evaluated in 2L STB (equipped with spin-filter) in order to reduce seed-train preparation time. PPRV were clarified using depth filtration (Sartopure PP2, Sartorius). Vero cells were adapted to ProVeroTM-1 SFM, reaching growth rates of 0.03 h-1 (similar to serum-containing cultures). The new in situ cell detachment method was successfully implemented, with yields above 80%; no impact on cell re-attachment or virus productivity was observed. A two-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining the new in situ cell detachment method with perfusion culture will enable the scale-up to 20L STB directly from a 2L STB, surpassing the need for a mid-scale platform and thus reducing seed-train preparation time. The potential of depth filtration for PPRV clarification (upon microcarriers sedimentation) could be confirmed, with yields up to 90%. Process scalability will be validated at the 20L scale in Sartorius BIOSTAT C-Plus (using engineering correlations such as shear stress and Kolmogorov-Eddy size as scale-up criteria) by comparing cells growth, metabolic and PPRV production kinetics to those achieved in 2L STB. In conclusion, the novel/scalable vaccine production process herein proposed has potential to assist the upcoming vaccination program for eradication of PPRV disease

Robust design and operation of quasi-continuous adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography

Adenoviruses (Ads) are considered one of the most suitable platforms for production of viral vaccines and gene therapy vectors. Their broad tissue tropism and large transgene packing capacity make them attractive candidates for innovative virotherapies. Ads can be produced in a complementary cell line in both adherent and suspension culture systems, such as HEK-293 or PER-C6 cells, or A549 for oncolytic therapies. Liquid chromatography is currently the core technique for vector purification, and its use is often integrated vertically within the downstream processing (DSP) strategy, as it easily fits into the early capture stage as well as into the final purification phase. The use of high-performance liquid chromatography (HPLC) for large-scale adenoviral purification is an already established method, including ion exchange (IEX), size exclusion (SEC), hydrophobic interaction, and immobilized metal affinity chromatography. Unlike traditional processes based on CsCl-gradient purification, HPLC offers a straightforward linear scale-up path, and procedures for purifying up to around 1014 input particles have been reported. SEC and ultra/diafiltration (usually by tangential flow filtration) are two other widely used processes at the very latest stage for formulating the product. One process-based way to improve the performance of the DSP chromatographic steps is by changing to a (quasi-)continuous processing mode, which, in principle, yields higher throughput, lower buffer consumption, higher capacity utilization of the stationary phase and reduced column volume, hence increased productivity. In this work, a simple, yet efficient, two-column simulated moving-bed (2CSMB) process for purifying adenovirus serotype 5 (Ad5) by size-exclusion chromatography (SEC) is presented and validated experimentally, and a general procedure for its robust design and operation under parameter uncertainty is described. The pilot-scale run yielded a virus recovery of 86% and DNA and HCP clearances of 90% and 89%, respectively, without any fine-tuning of the operating parameters. This performance compares very favorably against that of single-column batch chromatography for the same volume of size-exclusion resin. To improve the robustness of the 2CSMB-SEC process, a general procedure for its design under parameter uncertainty is proposed under the framework of rigorous optimization theory. The optimal values of the operating variables are chosen only among candidates that are robust feasible, that is, that remain feasible for all possible variations of the uncertain parameters within their uncertainty intervals. In practice the nominal design problem is replaced by a worst-case design problem. The procedure was successfully employed to find the optimal robust values of the step durations when both the interparticle porosity and column volume are subject to two types of uncertainties. In one case the parameters are uncorrelated, uniformly distributed random variables, whereas in the other case they are normally distributed random variables. Moreover, in the second case the 2CSMB-SEC operating conditions were optimized to yield a given probability of success of satisfying both product quality specifications, say 95%, so that on average at least 95 out of 100 attempts to pack the two columns yield purified batches in which the product is within the specifications—this is achieved while maximizing the feed throughput

Purification of a hepatitis C vaccine candidate: Comparison between multi- column chromatographic processes operated in positive and negative mode

Given the increasing efficiencies in bioreaction and growing interest on complex biopharmaceutical products such as virus-like particles (VLPs), downstream processing (DSP) is becoming ever more relevant. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields whilst improving product quality and lowering costs. One of most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. We report on the development and comparison of two types of multi-column chromatographic systems aimed at the purification of Hepatitis C VLPs, produced using insect cell-based expression with recombinant baculovirus. The first process described herein is based on direct product capture using an anion exchange chromatographic media and subsequent elution with the modulation of ionic strength. By using a multi-column approach, one is able to overcome the limits of dynamic binding capacity characteristic of single-column batch processes, thus increasing the media capacity utilization. The second process reported is based on negative chromatographic purification. In this approach elution conditions are such that impurities should adsorb on the chromatographic media whereas the product of interest flows through the column. Both process approaches are subjected to a temporal arrangement of operations steps suchlike column equilibration, product application, production and regeneration. Volumetric productivity thus depends not only on the optimal scheduling of the referred steps, but also upon factors such as media capacity for the product and related impurities, operational flow-rates, and mechanical limitations of the systems used. The proposed analysis compares volumetric productivity, resin capacity utilization, equipment footprint and skid complexity for both purification strategies. Also we will demonstrate that the optimal design is a balance between the manufacturing scale, complexity and imposed product quality requirements

Multi-column chromatographic purification of influenza virus-like particles

Seasonal Influenza occurs all over the world and causes approximately 500 million cases of infection and up to 500 thousand deaths annually. The most effective way to prevent the disease is throughout vaccination. However, the constant antigenic drift on the influenza virus implies an annually vaccine update with high inherent costs. A new generation of virus-like particles (VLPs) vaccines, that have the ability to stimulate the production of broad antibody response to different Influenza strains, is a promising approach to solve this problem. VLPs have become a promising solution for influenza pandemics as well. The high attractiveness of VLPs has led to an increasing interest in the development of VLP purification processes as it often accounts for the major production costs. Therefore, it is mandatory to improve downstream processing (DSP) trains, not only to increase the efficiency of the existing processes but also to develop new unit operations capable of coping with the stringent regulatory requirements. One of the most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography. The process herein described is based on the optimal scheduling of the operations steps characteristic of a single-column bind and elute operation such as equilibration, product application, production and regeneration applied to a train of two columns. In fact, a simple serial connection of two columns, during the product application step, directing the effluent of the first column in the train to an adjacent one, allows the capture of the mass transfer zone. This setup modification not only avoids product loss but also greatly increases the capacity utilization of the chromatographic media by achieving column saturation. We report on the development of a multi-column chromatographic process aimed at the purification of influenza VLPs, produced using insect cell-based expression with recombinant baculovirus. The inherent potential to improve process efficiency and economics, providing a powerful and flexible alternative to conventional batch chromatography will be demonstrated highlighting the impact of factors such as manufacturing scale, the complexity of the experimental setup and imposed product quality requirement

Process intensification for production of a peste des petites ruminants virus (PPRV) vaccine

Peste des Petites Ruminants Virus (PPRV) is a highly contagious disease affecting small ruminants in Africa and Asian countries, with negative/significant economic impact. Aiming to eradicate the disease, a novel/scalable PPRV vaccine production process is clearly needed. Built upon work previously done at iBET, a new production process is herein proposed using Vero cells growing on microcarriers, serum-free medium (SFM) and stirred-tank bioreactors (STB). This includes a new method for cells detachment from microcarriers, and perfusion culture for reducing turnaround time. The PPRV vaccine production process was developed in 2L STB (BIOSTAT DCU-3, Sartorius) using Nigeria 75/1 strain. Engineering correlations (e.g. shear stress and Eddy size) were used to optimize culture conditions. Vero cells were adapted to grow in ProVeroTM-1 SFM (Sartorius). A new enzymatic and mechanical method for in situ cell detachment from microcarriers was designed. PBS washing, TrypLE Select and trypsin inhibitor concentrations, and stirring rates were the variables explored. Perfusion culture was evaluated in 2L STB (equipped with spin-filter) in order to reduce seed-train preparation time. PPRV were clarified using depth filtration (Sartopure PP2, Sartorius). Vero cells were adapted to ProVeroTM-1 SFM, reaching growth rates of 0.03 h-1 (similar to serum-containing cultures). The new in situ cell detachment method was successfully implemented, with yields above 80%; no impact on cell re-attachment or virus productivity was observed. A two-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining the new in situ cell detachment method with perfusion culture will enable the scale-up to 20L STB directly from a 2L STB, surpassing the need for a mid-scale platform and thus reducing seed-train preparation time. The potential of depth filtration for PPRV clarification (upon microcarriers sedimentation) could be confirmed, with yields up to 90%. Process scalability will be validated at the 20L scale in Sartorius BIOSTAT C-Plus (using engineering correlations such as shear stress and Kolmogorov-Eddy size as scale-up criteria) by comparing cells growth, metabolic and PPRV production kinetics to those achieved in 2L STB. In conclusion, the novel/scalable vaccine production process herein proposed has potential to assist the upcoming vaccination program for eradication of PPRV disease

Advancing downstream purification of cell and gene therapy medicinal products

The advent of advanced therapies in the biopharmaceutical industry has moved the spotlight into complex products such as viral vectors or stem cells, holding great promise in a myriad of clinical targets. Currently, the challenge for a widespread application of these new biopharmaceuticals is the development of cost-effective bioprocesses while maintaining product\u27s bioactivity and quality attributes. This presentation will focus on the latest advancements on downstream purification of cell and gene therapy medicinal products, supported by process innovation and the flexibility of old, but robust technologies such as tangential flow filtration (TFF) and chromatography. Improvement of purification yields of virus based biopharmaceuticals can be achieved through the rational development of alternative strategies, combining different modes of operation, such as flow through purification or multi-column chromatography, together with the recent developments of chromatographic media and fundamental understanding of the adsorption phenomena as reported for the case of gene therapy medicinal products. Critical quality attributes of cell based medicinal products cannot be compromised by the processing route chosen. The use of the already established TFF technology has the potential to improve the purity of cell based products, with the evaluation of critical process parameters of cell concentration and washing being of paramount importance. The purity of such products can also be incremented with the use of negative mode expanded bed adsorption chromatography with a new multimodal prototype matrix based on core–shell bead technology as demonstrated for the case of human mesenchymal stem cells. In summary, the advancement of the purification of complex biopharmaceutical entities, such as the ones here reported, can be described as an incremental process, but still with space for inn