High-throughput Process Development Research Articles

High throughput screening setup of a scale-down device for membrane chromatography-aggregate removal of monoclonal antibodies.

In biopharmaceutical process development, resin-based high throughput screening (HTS) is well known for overcoming experimental limitations by permitting automated parallel processing at miniaturized scale, which results in fast data generation and reduced feed consumption. For membrane adsorber (MA), HTS solutions have so far only been available to a partial extent. Three case studies were performed with the aim of aligning HTS applications for MAs with those established for column chromatography: Process parameter range determination, mechanistic modeling (MM), and scalability. In order to exploit the MA typically features, such as high mass transfer and easy scalability, for scalable high throughput process development, a scale-down device (SDD) for MA was developed. Its applicability is confirmed for a monoclonal antibody aggregate removal step. The first case study explores the experimental application of the SDD developed. It uses bind and elute mode and variations of pH and salt concentration to obtain process operation windows for ion-exchange MAs Sartobind® S and Q. In the second case study, we successfully developed a mechanistic model based on parameters obtained from the SDD-HTS setup. The results proved to validate the use of the SDD developed for parameter estimation and thus model-based process development. The third case study shows the transferability and scalability of data from the SDD-HTS setup using both a direct scale factor and MM. Both approaches show good applicability with a deviation below 20% in the prediction of 10% dynamic breakthrough capacity and reliable scale-up from 0.42 to 800 ml.

Efficiency of Thermal Management Using Phase-Change Material for Nonisothermal Adsorption Process

Current energy issues have driven the development of high-throughput separation processes using solid adsorbents as an alternative to distillation. However, it is a crucial problem that the tempera...

A novel high-throughput process development screening tool for virus filtration

Virus filtration is used extensively in mammalian cell culture derived biopharmaceutical manufacturing processes. This processing step relies on bench scale models that require large volumes of filter load to demonstrate throughput, as well as expensive virus clearance studies to demonstrate clearance of virus spiked into load. A lack of adequate process knowledge about the virus clearance step can result in oversized virus filters and costly reprocessing steps. High throughput process development (HTPD) integrates parallel processing and automated scale-down models to minimize load volumes and process development timelines while maximizing process knowledge. Although a high throughput filter device would be useful for maximizing process knowledge on virus filtration step, a virus filter micro-scale HTPD model is not available. We designed a 96-well filter plate with a single layer of Viresolve® Pro filter media that was produced by MilliporeSigma (Burlington, MA USA). We tested the 96 well filter plate to assess its suitability to be a novel micro-scale HTPD scale-down model. In combination with an automated liquid handling robot, a Tecan EVO, our scale-down model generated flux decay curves for water, buffers and multiple different monoclonal antibodies (mAbs). The experimental setup demonstrated reproducibility and scale-up predictability. Virus filtration optimization is now a viable option using our HTPD scale-down model, enabling rapid process development using a fraction of the load material required for bench scale models while gaining a broader understanding of robustness of this important manufacturing step.

Acid-Free Electropolishing of Srf Nb Cavities

The International Linear Collider (ILC) is a 200–500 GeV center-of-mass linear electron-positron collider, based on 1.3 GHz superconducting radio-frequency accelerating technology.[1] The ILC will be used to gain a deeper understanding of the forces of energy and matter by colliding beams of electrons and positrons at nearly the speed of light. To achieve this goal ~16,000 RF superconducting cavities operating within two linear accelerators at near absolute zero are needed.[2] These SRF cavities are fabricated from pure Nb, which has an extremely low surface resistance at 2 Kelvin. To take full advantage of the Nb superconducting properties, the inner surface must be polished to a microscale roughness, and cleaned to be free of impurities that could degrade performance. The DOE seeks commercially viable, fabrication technologies for SRF cavities, specifically new or improved electropolishing technologies that do not use hydrofluoric acid. Current methods use high viscosity electrolytes containing hydrofluoric acid, which is not conducive to low-cost, high volume manufacturing and is potentially harmful to workers. Faraday is developing an electropolishing process for niobium SRF cavities, based on a new and evolving paradigm of non-viscous salt solution processing, enabled by a pulse reverse electric field as high-throughput vertical Acid-Free Final Electropolishing. The use of a low viscosity, near-neutral aqueous salt-based electrolyte enables a vertical cavity orientation, with no required rotation, which is conducive to high throughput manufacturing and industrialization. Furthermore, the use of this electrolyte combined with the pulse reverse or bipolar electric field changes the electropolishing mechanism by which material is removed and the surface profile reduced. Based on our limited understanding to date; [3] we have speculated that the process works via oxide film formation controlled during a designed anodic pulse, followed by an off time to remove heat and waste byproducts, followed by a cathodic pulse that removes the oxide film from the surface. This cycle is repeated many times per second, effectively removing niobium. The waveform design is such that the niobium is preferentially removed from the peaks on the surface, thus smoothing the surface. In addition, we will also discuss recent results from our development of high-throughput vertical acid-free final electropolishing process for SRF cavities and button cell cavity studies. The button cell cavity was designed and built by Cornell for their own cavity finishing process optimization using the conventional sulfuric acid HF approach. This tool enables rapid analysis of the effect of the electrofinishing applied parameter on the finish produced as a function of anode to cathode gap and active area ratio position of the button, orientation, gravity, electrofinishing conditions, and flow rate. Acknowledgements: Funding for this work is gratefully acknowledged from DOE SBIR Grant Number DE-SC0011235. [1] http://home.web.cern.ch/about/updates/2013/06/international-linear-collider-ready-construction[2] http://www.linearcollider.org/ILC/What-is-the-ILC/The-project[3] M. Inman et al “Electropolishing of Passive Materials in HF-Free Low Viscosity Aqueous Electrolytes, J. Electrochem. Soc., 160 (9) E94-E98 (2013).

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.

Development of microreactors with surface-immobilized biocatalysts for continuous transamination

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L−1 h−1, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.

Recent developments in chromatographic purification of biopharmaceuticals.

Over the last several decades, researchers have time and again proposed use of non-chromatographic methods for processing of biotherapeutic products. However, chromatography continues to be the backbone of downstream processing, particularly at process scale. There are many reasons for this, critical ones being the unparalleled scalability, robustness, and selectivity that process chromatography offers over its peers. It is no surprise then that process chromatography has been a topic of major developments in resin matrix, ligand chemistry, modalities, high throughput process development, process modelling, and approaches for control. In this review, we attempt to summarize major developments in the above-mentioned areas. Greater significance has been given to advancements in the last 5 years (2013-2017).

Optimization of a micro-scale, high throughput process development tool and the demonstration of comparable process performance and product quality with biopharmaceutical manufacturing processes

In this paper, we discuss the optimization and implementation of a high throughput process development (HTPD) tool that utilizes commercially available micro-liter sized column technology for the purification of multiple clinically significant monoclonal antibodies. Chromatographic profiles generated using this optimized tool are shown to overlay with comparable profiles from the conventional bench-scale and clinical manufacturing scale. Further, all product quality attributes measured are comparable across scales for the mAb purifications. In addition to supporting chromatography process development efforts (e.g., optimization screening), comparable product quality results at all scales makes this tool is an appropriate scale model to enable purification and product quality comparisons of HTPD bioreactors conditions. The ability to perform up to 8 chromatography purifications in parallel with reduced material requirements per run creates opportunities for gathering more process knowledge in less time.

High-Throughput Process Development for Biopharmaceuticals.

The ability to conduct multiple experiments in parallel significantly reduces the time that it takes to develop a manufacturing process for a biopharmaceutical. This is particularly significant before clinical entry, because process development and manufacturing are on the "critical path" for a drug candidate to enter clinical development. High-throughput process development (HTPD) methodologies can be similarly impactful during late-stage development, both for developing the final commercial process as well as for process characterization and scale-down validation activities that form a key component of the licensure filing package. This review examines the current state of the art for HTPD methodologies as they apply to cell culture, downstream purification, and analytical techniques. In addition, we provide a vision of how HTPD activities across all of these spaces can integrate to create a rapid process development engine that can accelerate biopharmaceutical drug development. Graphical Abstract.

High-throughput downstream process development for cell-based products using aqueous two-phase systems

As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research.

Rapid quantitation of monoclonal antibody N-glyco-occupancy and afucosylation using mass spectrometry

N-glyco-occupancy and afucoslyation level are two important quality attributes associated with N-glycosylation of therapeutic monoclonal antibodies (mAbs). We report here a fast mass spectrometry-based workflow for quantification of N-glycan site-occupancy and afucoslyation level of mAbs with improved throughput, precision, sensitivity and robustness. This method uses the deglycosylation after the first GlcNAc and inter-chain reduction of the mAbs, followed by liquid chromatography/mass spectrometry (LC-MS) analysis. The entire process can be completed within one hour, which provides a rapid quantitation of N-glyco-occupancy and afucosylation to support high-throughput cell line selection and process development for mAb biopharmaceuticals.

A scale-down cross-flow filtration technology for biopharmaceutical high throughput process development

Proteins are indispensable molecular machines present in all kingdoms of life, with exquisite diversity in structure and function arising from a small and simple set of building components called amino acids. Their participation in almost all biological reactions highlights their great importance to life itself and the pharmaceutical industry has also benefitted from their therapeutic worth. This chapter introduces the basic aspects for proteins, first discussing their biosynthesis from primary to quaternary structure, and then proceeding to discuss how they are able to carry out their function which is heavily dependent on their structure. We also discuss through illustration their involvement in industry given their role as therapeutics and as molecular targets for therapeutic development.

A robust robotic high-throughput antibody purification platform

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development.Miniaturization and automation of chromatography using micro columns (RoboColumns® from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO® from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH.The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A—desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches.

The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry.

A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity.

Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high-throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion-exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv-detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid-base titration. In contrast to acid-base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666-677, 2016.

Lessons learned in building high‐throughput process development capabilities

Companies have adapted high‐throughput techniques for process development to different levels. Some companies are beginning to incorporate high‐throughput process development (HTPD) techniques at a level which is approachable for the beginner user (example: plate‐based resin screening for purification) and others have sophisticated robotic platforms which are in routine use for new pipeline products for upstream, downstream, analytics, and just now starting in the protein formulation area. This mini review serves to summarize how companies have set up and are using HTPD capabilities, including the need for user‐groups to improve the likelihood of success. The focus will be a practical summary of the utility and limitations of these approaches.

High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography

The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles.

High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli

The production of safe vaccines against untreatable or new diseases has pushed the research in the field of virus-like particles (VLPs). Currently, a large number of commercial VLP-based human vaccines and vaccine candidates are available or under development. A promising VLP production route is the controlled in vitro assembly of virus proteins into capsids.In the study reported here, a high-throughput screening (HTS) procedure was implemented for the upstream process development of a VLP platform in bacterial cell systems. Miniaturized cultivations were carried out in 48-well format in the BioLector system (m2p-Labs, Germany) using an Escherichia coli strain with a tac promoter producing the murine polyomavirus capsid protein (VP1). The screening procedure incorporated micro-scale cultivations, HTS cell disruption by sonication and HTS-compatible analytics by capillary gel electrophoresis. Cultivation temperatures, shaking speeds, induction and medium conditions were varied to optimize the product expression in E. coli. The most efficient system was selected based on an evaluation of soluble and insoluble product concentrations as well as on the percentage of product in the total soluble protein fraction. The optimized system was scaled up to cultivation 2.5L shaker flask scale and purified using an anion exchange chromatography membrane adsorber, followed by a size exclusion chromatography polishing procedure. For proof of concept, purified VP1 capsomeres were assembled under defined buffer conditions into empty capsids and characterized using transmission electron microscopy (TEM).The presented HTS procedure allowed for a fast development of an efficient production process of VLPs in E. coli. Under optimized cultivation conditions, the VP1 product totalled up to 43% of the total soluble protein fraction, yielding 1.63mg VP1 per mL of applied cultivation medium. The developed production process strongly promotes the murine polyoma-VLP platform, moving towards an industrially feasible technology for new chimeric vaccines.

High‐throughput process development: Chromatography media volume definition

Prediction of chromatographic separations in liter scale with the help of data gathered using a microliter format is challenging, as both scale effects and differences in the formats used (plates vs. packed minicolumns) need to be addressed. The use of microplates in high‐throughput process development (HTPD) studies has become a routine. While this method is typically used for investigating chromatography conditions such as salt and pH, it can also be used for screening of different chromatography media (resins). In this study, the influence of the physicochemical properties of various media on preparation of medium‐containing microplates is discussed, and examples are presented that show how these properties can influence the conclusions drawn in benchmarking studies done using this HTPD format. The results indicate that differences in chromatographic media volume between microliter sampling techniques and packed chromatography columns do exist. Regardless of the chosen chromatography media, sampling technique or volume definition method, the simplest way to address this issue is by calibrating the microliter scale with packed‐column volumes in appropriate dry weight experiments.

An integrated robotic system for high‐throughput process development of cell and virus culture conditions: Application to biosafety level 2 live virus vaccines

Live virus vaccines are a critical component of worldwide vaccination strategy for reducing disease burden but often require complex biological production processes that are sensitive to many different factors, both known and often unknown. Prior application of high‐throughput process development (HTPD) approaches to these processes has been hampered by a complex design space, low‐throughput analytics, and challenges inherent in biosafety level 2 containment and asepsis in laboratory automation. In 2013, we initiated a project with HighRes Biosolutions to design and install an integrated high‐throughput screening platform to enable HTPD for biosafety level 2 upstream process development studies. The system incorporates the necessary tools for performing cell and virus culture studies in microplates, as well as advanced analytical capabilities necessary for assessment of cell phenotype, product quality, and antigen yield. To date, we have applied this system to screen optimal media formulations and viral production conditions in support of two viral vaccine programs, with phenotypic assays performed as an integrated part of the workflow. This case study illustrates the power of HTPD in addressing large‐scale biological screening challenges by narrowing a vast design space and identifying parameter interactions in live virus production processes.