Autologous CAR T-cell therapies supply chain: challenges and opportunities?

Chimeric antigen receptor (CAR) T cells are considered a potentially disruptive cancer therapy, showing highly promising results. Their recent success and regulatory approval (both in the USA and Europe) are likely to generate a rapidly increasing demand and a need for the design of robust and scalable manufacturing and distribution models that will ensure timely and cost-effective delivery of the therapy to the patient. However, there are challenging tasks as these therapies are accompanied by a series of constraints and particularities that need to be taken into consideration in the decision-making process. Here, we present an overview of the current state of the art in the CAR T cell market and present novel concepts that can debottleneck key elements of the current supply chain model and, we believe, help this technology achieve its long-term potential.

High throughput process development workflow with advanced decision-support for antibody purification.

Chromatography remains the workhorse in antibody purification; however process development and characterisation still require significant resources. The high number of operating parameters involved requires extensive experimentation, traditionally performed at small- and pilot-scale, leading to demands in terms of materials and time that can be a challenge. The main objective of this research was the establishment of a novel High Throughput Process Development (HTPD) workflow combining scale-down chromatography experimentation with advanced decision-support techniques in order to minimise the consumption of resources and accelerate the development timeframe. Additionally, the HTPD workflow provides a framework to rapidly manipulate large datasets in an automated fashion. The central component of the HTPD workflow is the systematic integration of a microscale chromatography experimentation strategy with an advanced chromatogram evaluation method, design of experiments (DoE) and multivariate data analysis. The outputs of this are leveraged into the screening and optimisation components of the workflow. For the screening component, a decision-support tool was developed combining different multi-criteria decision-making techniques to enable a fair comparison of a number of CEX resin candidates and determine those that demonstrate superior purification performance. This provided a rational methodology for screening chromatography resins and process parameters. For the optimisation component, the workflow leverages insights provided through screening experimentation to guide subsequent DoE experiments so as to tune significant process parameters for the selected resin. The resulting empirical correlations are linked to a stochastic modelling technique so as to predict the optimal and most robust chromatographic process parameters to achieve the desired performance criteria.

Data-driven multi-objective optimization via grid compatible simplex technique and desirability approach for challenging high throughput chromatography applications.

Recently, a grid compatible Simplex variant has been demonstrated to identify optima consistently and rapidly in challenging high throughput (HT) applications in early bioprocess development. Here, this method is extended by deploying it to multi-objective optimization problems. Three HT chromatography case studies are presented, each posing challenging early development situations and including three responses which were amalgamated by the adoption of the desirability approach. The suitability of a design of experiments (DoE) methodology per case study, using regression analysis in addition to the desirability approach, was evaluated for a large number of weights and in the presence of stringent and lenient performance requirements. Despite the adoption of high-order models, this approach had low success in identification of the optimal conditions. For the deployment of the Simplex approach, the deterministic specification of the weights of the merged responses was avoided by including them as inputs in the formulated multi-objective optimization problem, facilitating this way the decision making process. This, and the ability of the Simplex method to locate optima, rendered the presented approach highly successful in delivering rapidly operating conditions, which belonged to the Pareto set and offered a superior and balanced performance across all outputs compared to alternatives. Moreover, its performance was relatively independent of the starting conditions and required sub-minute computations despite its higher order mathematical functionality compared to DoE techniques. These evidences support the suitability of the grid compatible Simplex method for early bioprocess development studies involving complex data trends over multiple responses. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:1393-1406, 2018.

Economic evaluation of the primary recovery of tetracycline with traditional and novel aqueous two-phase systems.

Antibiotics are a key pharmaceutical to inhibit growth or kill microorganisms. They represent a profitable market and, in particular, tetracycline has been listed as an essential medicine by the WHO. Therefore it is important to improve their production processes. Recently novel and traditional aqueous two-phase systems for the extraction have been developed with positive results. The present work performs an economic analysis of the production and recovery of tetracycline through the use of several ATPS through bioprocess modeling using specialized software (BioSolve, Biopharm Services Ltd, UK) to determine production costs per gram (CoG/g). First, a virtual model was constructed using published data on the recovery of tetracycline and extended to incorporate uncertainties. To determine how the model behaved, a sensitivity analysis and Monte Carlo simulations were performed. Results showed that ATPS formed by cholinium chloride/K3PO4 was the best option to recover tetracycline, as it had the lowest CoG/g (US$ 672.83/g), offered the highest recovery yield (92.42%), second best sample input capacity (45% of the ATPS composition) and one of the lowest materials contribution to cost. The ionic liquid-based method of ATPS is a promising alternative for recovering tetracycline from fermentation broth.

Effects of bed compression on protein separation on gel filtration chromatography at bench and pilot scale.

BACKGROUND: Poorly packed chromatography columns are known to reduce drastically the column efficiency and produce broader peaks. Controlled bed compression has been suggested to be a useful approach for solving this problem. Here the relationship between column efficiency and resolution of protein separation are examined when preparative chromatography media were compressed using mechanical and hydrodynamic methods. Sepharose CL-6B, an agarose based size exclusion media was examined at bench and pilot scale. The asymmetry and height equivalent of a theoretical plate (HETP) was determined by using 2% v/v acetone, whereas the void volume and intraparticle porosity (ϵ p) were estimated by using blue dextran. A protein mixture of ovalbumin (chicken), bovine serum albumin (BSA) and γ'- globulin (bovine) with molecular weights of 44, 67 and 158 kDa, respectively, were used as a 'model' separation challenge. RESULTS: Mechanical compression achieved a reduction in plate height for the column with a concomitant improvement in asymmetry. Furthermore, the theoretical plate height decreased significantly with mechanical compression resulting in a 40% improvement in purity compared with uncompressed columns at the most extreme conditions of compression used. CONCLUSION: The results suggest that the mechanical bed compression of Sepharose CL-6B can be used to improve the resolution of protein separation. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies.

Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form of filtration, centrifugation, or a combination of both for cell separation and/or retention. Filtration methods include normal flow depth filtration and tangential flow microfiltration. The ability to scale down predictably the selected harvest method helps to ensure successful production and is critical for conducting small-scale characterization studies for confirming parameter targets and ranges. In this chapter we describe centrifugation and depth filtration harvesting methods, share strategies for harvest optimization, present recent developments in centrifugation scale-down models, and review alternative harvesting technologies.

Estimating preferences for modes of drug administration: The case of US healthcare professionals.

BACKGROUND: There are hidden drug administration costs that arise from a mismatch between end-user preferences and how manufacturers choose to formulate their drug products for delivery to patients. The corollary of this is: there are "intangible benefits" from considering end-user preferences in manufacturing patient-friendly medicines. It is important then to have some idea of what pharmaceutical manufacturers should consider in making patient-friendly medicines and of the magnitude of the indirect benefits from doing so. OBJECTIVES: This study aimed to evaluate preferences of healthcare professionals in the US for the non-monetary attributes of different modes of drug administration. It uses these preference orderings to compute a monetary valuation of the indirect benefits from making patient-friendly medicines. METHODS: A survey collected choice preferences of a sample of 210 healthcare professionals in the US for two unlabelled drug options. These drugs were identical except in the levels of attributes of drug administration. Using the choice data collected, statistical models were estimated to compute gross welfare benefits, measured by the expected compensating variation, from making drugs in a more patient-friendly manner. RESULTS: The monetary value of end-user benefits from developing patient-friendly drug delivery systems is: (1) as large as the annual acquisition costs per full treatment episode for some biologic drugs; and (2) likely to fall in the "high end" of the distribution of the direct monetary costs of drug administration. CONCLUSIONS: An examination of end-user preferences should help manufacturers make more effective and efficient use of limited resources for innovations in drug delivery system, or manufacturing research in general.

Simplex-based optimization of numerical and categorical inputs in early bioprocess development: Case studies in HT chromatography.

Bioprocess development studies often involve the investigation of numerical and categorical inputs via the adoption of Design of Experiments (DoE) techniques. An attractive alternative is the deployment of a grid compatible Simplex variant which has been shown to yield optima rapidly and consistently. In this work, the method is combined with dummy variables and it is deployed in three case studies wherein spaces are comprised of both categorical and numerical inputs, a situation intractable by traditional Simplex methods. The first study employs in silico data and lays out the dummy variable methodology. The latter two employ experimental data from chromatography based studies performed with the filter-plate and miniature column High Throughput (HT) techniques. The solute of interest in the former case study was a monoclonal antibody whereas the latter dealt with the separation of a binary system of model proteins. The implemented approach prevented the stranding of the Simplex method at local optima, due to the arbitrary handling of the categorical inputs, and allowed for the concurrent optimization of numerical and categorical, multilevel and/or dichotomous, inputs. The deployment of the Simplex method, combined with dummy variables, was therefore entirely successful in identifying and characterizing global optima in all three case studies. The Simplex-based method was further shown to be of equivalent efficiency to a DoE-based approach, represented here by D-Optimal designs. Such an approach failed, however, to both capture trends and identify optima, and led to poor operating conditions. It is suggested that the Simplex-variant is suited to development activities involving numerical and categorical inputs in early bioprocess development.

Optimization-based framework for resin selection strategies in biopharmaceutical purification process development.

This work addresses rapid resin selection for integrated chromatographic separations when conducted as part of a high-throughput screening exercise during the early stages of purification process development. An optimization-based decision support framework is proposed to process the data generated from microscale experiments to identify the best resins to maximize key performance metrics for a biopharmaceutical manufacturing process, such as yield and purity. A multiobjective mixed integer nonlinear programming model is developed and solved using the ε-constraint method. Dinkelbach's algorithm is used to solve the resulting mixed integer linear fractional programming model. The proposed framework is successfully applied to an industrial case study of a process to purify recombinant Fc Fusion protein from low molecular weight and high molecular weight product related impurities, involving two chromatographic steps with eight and three candidate resins for each step, respectively. The computational results show the advantage of the proposed framework in terms of computational efficiency and flexibility. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1116-1126, 2017.

An automated laboratory-scale methodology for the generation of sheared mammalian cell culture samples.

Continuous disk-stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing-scale. The use of scale-down methods to characterise disk-stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot-scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy-to-assemble automated capillary-based methodology to generate levels of EDRs consistent with those found in a continuous disk-stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot-scale centrifuge using the experimental method developed in this paper (2.4×105 W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×105 W/Kg). Furthermore, this methodology can be incorporated into existing scale-down methods to model the process performance of continuous disk-stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality.

Life-cycle and cost of goods assessment of fed-batch and perfusion-based manufacturing processes for mAbs.

Life-cycle assessment (LCA) is an environmental assessment tool that quantifies the environmental impact associated with a product or a process (e.g., water consumption, energy requirements, and solid waste generation). While LCA is a standard approach in many commercial industries, its application has not been exploited widely in the bioprocessing sector. To contribute toward the design of more cost-efficient, robust and environmentally-friendly manufacturing process for monoclonal antibodies (mAbs), a framework consisting of an LCA and economic analysis combined with a sensitivity analysis of manufacturing process parameters and a production scale-up study is presented. The efficiency of the framework is demonstrated using a comparative study of the two most commonly used upstream configurations for mAb manufacture, namely fed-batch (FB) and perfusion-based processes. Results obtained by the framework are presented using a range of visualization tools, and indicate that a standard perfusion process (with a pooling duration of 4 days) has similar cost of goods than a FB process but a larger environmental footprint because it consumed 35% more water, demanded 17% more energy, and emitted 17% more CO2 than the FB process. Water consumption was the most important impact category, especially when scaling-up the processes, as energy was required to produce process water and water-for-injection, while CO2 was emitted from energy generation. The sensitivity analysis revealed that the perfusion process can be made more environmentally-friendly than the FB process if the pooling duration is extended to 8 days. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1324-1335, 2016.

Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures.

Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost-effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale-down (USD) mimics requiring 25-110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost-effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario.

A scale-down mimic for mapping the process performance of centrifugation, depth and sterile filtration.

In the production of biopharmaceuticals disk-stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot-scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale-down approach based upon the use of a shear device and a bench-top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large-scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934-1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle).

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques.

IMAC capture of recombinant protein from unclarified mammalian cell feed streams.

Fusion-tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300-500 µm diameter agarose resin beads that allow free passage of cells but capture His-tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His-tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼ 8 U/mL and 2 ng/µL in column flow-through, respectively. Recovery of His-tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams.

High throughput screening of particle conditioning operations: I. System design and method development.

The biotech industry is under increasing pressure to decrease both time to market and development costs. Simultaneously, regulators are expecting increased process understanding. High throughput process development (HTPD) employs small volumes, parallel processing, and high throughput analytics to reduce development costs and speed the development of novel therapeutics. As such, HTPD is increasingly viewed as integral to improving developmental productivity and deepening process understanding. Particle conditioning steps such as precipitation and flocculation may be used to aid the recovery and purification of biological products. In this first part of two articles, we describe an ultra scale-down system (USD) for high throughput particle conditioning (HTPC) composed of off-the-shelf components. The apparatus is comprised of a temperature-controlled microplate with magnetically driven stirrers and integrated with a Tecan liquid handling robot. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. A comprehensive suite of high throughput analytics enables measurement of product titer, product quality, impurity clearance, clarification efficiency, and particle characterization. HTPC at the 1 mL scale was evaluated with fermentation broth containing a vaccine polysaccharide. The response profile was compared with the Pilot-scale performance of a non-geometrically similar, 3 L reactor. An engineering characterization of the reactors and scale-up context examines theoretical considerations for comparing this USD system with larger scale stirred reactors. In the second paper, we will explore application of this system to industrially relevant vaccines and test different scale-up heuristics.

High throughput screening of particle conditioning operations: II. Evaluation of scale-up heuristics with prokaryotically expressed polysaccharide vaccines.

Multivalent polysaccharide conjugate vaccines are typically comprised of several different polysaccharides produced with distinct and complex production processes. Particle conditioning steps, such as precipitation and flocculation, may be used to aid the recovery and purification of such microbial vaccine products. An ultra scale-down approach to purify vaccine polysaccharides at the micro-scale would greatly enhance productivity, robustness, and speed the development of novel conjugate vaccines. In part one of this series, we described a modular and high throughput approach to develop particle conditioning processes (HTPC) for biologicals that combines flocculation, solids removal, and streamlined analytics. In this second part of the series, we applied HTPC to industrially relevant feedstreams comprised of capsular polysaccharides (CPS) from several bacterial species. The scalability of HTPC was evaluated between 0.8 mL and 13 L scales, with several different scaling methodologies examined. Clarification, polysaccharide yield, impurity clearance, and product quality achieved with HTPC were reproducible and comparable with larger scales. Particle sizing was the response with greatest sensitivity to differences in processing scale and enabled the identification of useful scaling rules. Scaling with constant impeller tip speed or power per volume in the impeller swept zone offered the most accurate scale up, with evidence that time integration of these values provided the optimal basis for scaling. The capability to develop a process at the micro-scale combined with evidence-based scaling metrics provide a significant advance for purification process development of vaccine processes. The USD system offers similar opportunities for HTPC of proteins and other complex biological molecules.

Economic analysis of Royalactin production under uncertainty: Evaluating the effect of parameter optimization.

Royalactin is a protein with several different potential uses in humans. Research, in insects and in mammalian cells, has shown that it can accelerate cell division and prevent apoptosis. The method of action is through the use of the epidermal growth factor receptor, which is present in humans. Potential use in humans could be to lower cholesterolemic levels in blood, and to elicit similar effects to those seen in bees, e.g., increased lifespan. Mass production of Royalactin has not been accomplished, though a recent article presented a Pichia pastoris fermentation and recovery by aqueous two-phase systems at laboratory scale as a possible basis for production. Economic modelling is a useful tool with which compare possible outcomes for the production of such a molecule and in particular, to locate areas where additional research is needed and optimization may be required. This study uses the BioSolve software to perform an economic analysis on the scale-up of the putative process for Royalactin. The key parameters affecting the cost of production were located via a sensitivity analysis and then evaluated by Monte Carlo analysis. Results show that if titer is not optimized the strategy to maintain a low cost of goods is process oriented. After optimization of this parameter the strategy changes to a product-oriented and the target output becomes the critical parameter determining the cost of goods. This study serves to provide a framework for the evaluation of strategies for future production of Royalactin, by analyzing the factors that influence its cost of manufacture.

Representative mammalian cell culture test materials for assessment of primary recovery technologies: a rapid method with industrial applicability.

Mammalian cell culture material is often difficult to produce accurately and reproducibly for downstream studies. This article presents a methodology for the creation of a set of cell culture test materials where key variables including cell density, cell viability, product, and the host cell protein (HCP) load can be manipulated individually. The methodology was developed using a glutamine synthetase Chinese hamster ovary cell line cultured at 5-L and 70-L scales. Cell concentration post-cell growth was manipulated using tangential flow filtration to generate a range of target cell densities of up to 100 × 10(6) cells/mL. A method to prepare an apoptotic cell stock to achieve target viabilities of 40-90% is also described. In addition, a range of IgG1 and HCP concentrations was achieved. The results illustrate that the proposed methodology is able to mimic different cell culture profiles by decoupling the control of the key variables. The cell culture test materials were shown to be representative of typical cell culture feed material in terms of particle size distribution and HCP population. This provides a rapid method to create the required feeds for assessing the feasibility of primary recovery technologies designed to cope with higher cell density cultures.