High-throughput Process Development Research Articles

Integrated micro-scale protein a chromatography and Low pH viral inactivation unit operations on an automated platform.

High throughput process development (HTPD) is established for time- and resource- efficient chromatographic process development. However, integration with non-chromatographic operations within a monoclonal antibody (mAb) purification train is less developed. An area of importance is the development of low pH viral inactivation (VI) that follows protein A chromatography. However, the lack of pH measurement devices at the micro-scale represents a barrier to implementation, which prevents integration with the surrounding unit operations, limiting overall process knowledge. This study is based upon the design and testing of a HTPD platform for integration of the protein A and low pH VI operations. This was achieved by using a design and simulation software before execution on an automated liquid handler. The operations were successfully translated to the micro-scale, as assessed by analysis of recoveries and molecular weight content. The integrated platform was then used as a tool to assess the effect of pH on HMWC during low pH hold. The laboratory-scale and micro-scale elution pools showed comparable HMWC across the pH range 3.2-3.7. The investigative power of the platform is highlighted by evaluating the resources required to conduct a hypothetical experiment. This results in lower resource demands and increased labor efficiency relative to the laboratory-scale. For example, the experiment can be conducted in 7 h, compared to 105 h, translating to labor hours, 3 h and 28 h for the micro-scale and laboratory-scale, respectively. This presents the opportunity for further integration beyond chromatographic operations within the purification sequence, to establish a fit-to-platform assessment tool for mAb process development.

Digital twin in high throughput chromatographic process development for monoclonal antibodies

The monoclonal antibody (mAb) industry is becoming increasingly digitalized. Digital twins are becoming increasingly important to test or validate processes before manufacturing. High-Throughput Process Development (HTPD) has been progressively used as a tool for process development and innovation. The combination of High-Throughput Screening with fast computational methods allows to study processes in-silico in a fast and efficient manner. This paper presents a hybrid approach for HTPD where equal importance is given to experimental, computational and decision-making stages. Equilibrium adsorption isotherms of 13 protein A and 16 Cation-Exchange resins were determined with pure mAb. The influence of other components in the clarified cell culture supernatant (harvest) has been under-investigated. This work contributes with a methodology for the study of equilibrium adsorption of mAb in harvest to different protein A resins and compares the adsorption behavior with the pure sample experiments. Column chromatography was modelled using a Lumped Kinetic Model, with an overall mass transfer coefficient parameter (kov). The screening results showed that the harvest solution had virtually no influence on the adsorption behavior of mAb to the different protein A resins tested. kov was found to have a linear correlation with the sample feed concentration, which is in line with mass transfer theory. The hybrid approach for HTPD presented highlights the roles of the computational, experimental, and decision-making stages in process development, and how it can be implemented to develop a chromatographic process. The proposed white-box digital twin helps to accelerate chromatographic process development.

Model-based control system design to manage process parameters in mammalian cell culture for biopharmaceutical manufacturing.

To enhance the robustness and flexibility of biopharmaceutical manufacturing, a paradigm shift toward methods of continuous processing, such as perfusion, and fundamental technologies for high-throughput process development are being actively investigated. The continuous upstream process must establish an advanced control strategy to ensure a "State of Control" before operation. Specifically, feedforward and feedback control must address the complex fluctuations that occur during the culture process and maintain critical process parameters in appropriate states. However, control system design for industry-standard mammalian cell culture processes is still often performed in a laborious trial-and-error manner. This paper provides a novel control approach in which controller specifications to obtain desired control characteristics can be determined systematically by combining a culture model with control theory. In the proposed scheme, control conditions, such as PID parameters, can be specified mechanistically based on process understanding and control requirements without qualitative decision making or specific preliminary experiments. The effectiveness of the model-based control algorithm was verified by control simulations assuming perfusion Chinese hamster ovary culture. As a tool to assist in the development of control strategies, this study will reduce the high operational workload that is a serious problem in continuous culture and facilitate the digitalization of bioprocesses.

Development of high-throughput rapid heat-treatment and characterization process

Heat treatment plays an important part in materials development, but traditional heat treatment methods can only develop one target temperature/cycle at a time, which impairs the experimental efficiency in the development of new materials. We have therefore introduced the use of high-throughput experiments, changed the traditional sequential iterative method to parallel processing, and developed an efficient multi-temperature-field heat-treatment method. Microwave radiation is used as the heating source, and the method takes advantage of the selective heating ability of microwaves to generate 100 temperature zones that can heat treat 100 individual sheet samples simultaneously. The method incorporates various rapid characterization techniques, which enables a large amount of characterization data to be obtained in a short time. The obtained data can be uploaded to a high-throughput materials database to provide data to support subsequent studies. In this study, high-carbon, high-chromium steel was subjected to parallel tempering heat treatment at 109–786 °C. The relationship between the tempering temperature and the material microstructure and properties was derived from the changes in the microstructure and properties after parallel tempering, and used to verify the feasibility of this method.

A comprehensive assessment of the applicability of RoboColumn as a chromatography scale-down model for use in biopharmaceutical process validation

High-throughput process development has become a standard practice in the biopharmaceutical industry to enable time, cost, and material savings. In downstream biopharmaceutical process development, miniaturized, parallelized chromatography columns, known as RoboColumn, have become the standard for process development, as RoboColumn have shown generally comparable performance to bench and manufacturing scale columns. However, RoboColumn have yet to be widely implemented in process validation and characterization, where many multifactor experiments are typically executed, and there is a strong value proposition for performing high-throughput experiments. The hesitancy to utilize RoboColumn in process validation arises from scale differences that result in exacerbated peak broadening at RoboColumn scale relative to traditional bench or manufacturing scales. Thus, to support reliable application of RoboColumn in process validation, the present study provides a comprehensive investigation to understand how scale differences affect chromatographic performance by comparing RoboColumn, bench, and manufacturing scales using seven different production processes covering three different antibody formats, five different resin types, and three chromatographic modes of operation. RoboColumn chromatographic performance was compared at target and off-target conditions to emulate scale-down model qualification and multifactor studies, respectively. RoboColumn demonstrated good comparability at both target and off-target process conditions. To further demonstrate an understanding of comparability, a study was performed to show a rare case in which product quality offsets may occur as a result RoboColumn scale differences. By showing scale comparability and an understanding of potential offsets, this work demonstrates that RoboColumn can be used in any stage of process development, including process validation and characterization.

Automated calibration and in-line measurement of product quality during therapeutic monoclonal antibody purification using Raman spectroscopy.

Current manufacturing and development processes for therapeutic monoclonal antibodies demand increasing volumes of analytical testing for both real-time process controls and high-throughput process development. The feasibility of using Raman spectroscopy as an in-line product quality measuring tool has been recently demonstrated and promises to relieve this analytical bottleneck. Here, we resolve time-consuming calibration process that requires fractionation and preparative experiments covering variations of product quality attributes (PQAs) by engineering an automation system capable of collecting Raman spectra on the order of hundreds of calibration points from two to three stock seed solutions differing in protein concentration and aggregate level using controlled mixing. We used this automated system to calibrate multi-PQA models that accurately measured product concentration and aggregation every 9.3 s using an in-line flow-cell. We demonstrate the application of a nonlinear calibration model for monitoring product quality in real-time during a biopharmaceutical purification process intended for clinical and commercial manufacturing. These results demonstrate potential feasibility to implement quality monitoring during GGMP manufacturing as well as to increase chemistry, manufacturing, and controls understanding during process development, ultimately leading to more robust and controlled manufacturing processes.

Protein A Chromatography Purification for a Monoclonal Antibody from Process Development to Scale-up

Monoclonal antibodies have been established as a major product class of biotechnology-based drugs. The increasing demand of monoclonal antibodies has led pharmaceutical companies to adopt efficient production processes. Transferring monoclonal antibody production to the industrial manufacturing requires adequate effort in process development. The strategy to reduce development time and cost comprises high-throughput process development which is especially central for the rapid optimization of the purification process. Chromatography process is the backbone of the purification process that can deliver high purity but it requires significant resources. Combined with high-throughput process development approach, the chromatography process is easy to develop and scale-up from laboratory to manufacturing scale. Design of experiments helps high-throughput process development workflow to provide decision-support techniques. This approach ensures significantly decreased time and material needs while improving the chromatography process. Protein A affinity chromatography is one of the most important chromatographic steps because of its great performance and capabilities. Most of the working parameters can be predefined and are identical for several monoclonal antibodies. However, some parameters like elution pH, loading capacity, resin type need further optimization for each monoclonal antibody. In this study, the loading and elution parameters were screened for Protein A chromatography to identify the best purification conditions using the combination of Design of Experiments and high-throughput process development approach in micro-volume columns. Developed working parameters were used for scale-up and tested under robust process conditions. Specific chromatography conditions were applied in pilot-scale and data comparison was done with micro-volume columns, lab-column scale to validate high-throughput strategy approach

Demonstration of continuous gradient elution functionality with automated liquid handling systems for high-throughput purification process development

Various high-throughput systems and strategies are employed by the biopharmaceutical industry for early to late-stage process development for biologics manufacturing. The associated increases to experiment productivity and reduction in material consumption makes high throughput tools integral for bioprocess development. While these high-throughput systems have been successfully leveraged to generate high quality data representative of manufacturing scale processes, their data interpretation often requires complex data transformation and time-intensive system characterization. With respect to high throughput purification development, RoboColumns by Repligen operated on Tecan automated liquid handling systems offer superior performance scalability, but lack an optimized liquid delivery system that is representative of preparative chromatography. Particularly, stock Tecan liquid handling systems lack the capability to provide high-capacity continuous liquid flow and ideal linear gradient chromatography conditions. These limitations impact protein chromatography performance and hinder the application of high-throughput gradient elution experiments. In this work, we describe a Tecan Freedom EVO high-throughput purification tool that provides more continuous liquid delivery enabling continuous gradient elution capability for RoboColumn experiments as demonstrated by generation of highly linear conductivity gradients. Results demonstrate that the tool can provide RoboColumn performance and product quality data that is in agreement with larger, bench scale chromatography formats for two model purification methods. The described gradient purification method also provides more consistent performance between RoboColumns and larger column formats compared to step elution methods using the same optimized Tecan system. Lastly, new insights into the impact of discontinuous flow on RoboColumn elution performance are introduced, which may help further improve application of these data towards bioprocess development.

Recent advances to accelerate purification process development: A review with a focus on vaccines

The safety requirements for vaccines are extremely high since they are administered to healthy people. For that reason, vaccine development is time-consuming and very expensive. Reducing time-to-market is key for pharmaceutical companies, saving lives and money. Therefore the need is raised for systematic, general and efficient process development strategies to shorten development times and enhance process understanding. High throughput technologies tremendously increased the volume of process-related data available and, combined with statistical and mechanistic modeling, new high throughput process development (HTPD) approaches evolved. The introduction of model-based HTPD enabled faster and broader screening of conditions, and furthermore increased knowledge. Model-based HTPD has particularly been important for chromatography, which is a crucial separation technique to attain high purities. This review provides an overview of downstream process development strategies and tools used within the (bio)pharmaceutical industry, focusing attention on (protein subunit) vaccine purification processes. Subsequently high throughput process development and other combinatorial approaches are discussed and compared according to their experimental effort and understanding. Within a growing sea of information, novel modeling tools and artificial intelligence (AI) gain importance for finding patterns behind the data and thereby acquiring a deeper process understanding.

High-throughput multiplex analysis of mAb aggregates and charge variants by automated two-dimensional size exclusion-cation exchange chromatography coupled to mass spectrometry

Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis.

Demonstration of Continuous Gradient Elution Functionality with Automated Liquid Handling Systems for High-Throughput Purification Process Development

Demonstration of Continuous Gradient Elution Functionality with Automated Liquid Handling Systems for High-Throughput Purification Process Development

High throughput chromatography and analytics can inform viral clearance capabilities during downstream process development for biologics.

High throughput process development (HTPD) using liquid handling robotics and RoboColumns is an established methodology in downstream process development to screen chromatography resins and optimize process designs to meet target product profiles. However, HTPD is not yet widely available for use in viral clearance capability of the resin due to a variety of constraints. In the present study, a BSL-1-compatible, non-infectious MVM model, MVM-VLP, was tested for viral clearance assessment with various resin and membrane chromatography operations in a HTPD mode. To detect the MVM-VLP in the high throughput experiments, an electrochemiluminescence immunoassay (ECLIA) assay was developed with up to 5 logs of dynamic range. Storage time suitability of MVM-VLP solutions in various buffer matrices, in the presence or absence of a glycoprotein vaccine candidate, were assessed. Then, MVM-VLP and a test article monoclonal antibody (mAb) were used in a HTPD design that included commercially available ion exchange media chemistries, elucidating a wide variety of viral clearance ability at different operating conditions. The methodologies described herein have the potential to be a part of the process design stage in biologics manufacturing process development, which in turn can reduce risk associated with viral clearance validation studies.

Automation and miniaturization: enabling tools for fast, high‐throughput process development in integrated continuous biomanufacturing

AbstractProcess development in the biotech industry leads to investments around hundred of millions of dollars. It is important to mitigate costs without neglecting the quality of process development. Biopharmaceutical process development is important for companies to develop new processes and be first to market, improve a pre‐established process, or start manufacturing a product available by patent expiry (biosimilars). Laboratory automation enables methodical and standardized process development. Miniaturization and parallelization empower laboratories to screen several experimental conditions and define operating windows for purification processes, improving process robustness. Together, they allow for fast and accurate process development in a fraction of the time and cost of nonminiaturized/nonparallel process development approaches. The most widely used High‐Throughput Screening technique is a liquid‐handling station and microfluidics is taking its first steps in process development. Both are attractive scale‐down tools for the characterization of bioprocesses and allow thousands of experiments to be performed per day. High‐Throughput Process Development (HTPD) has helped to achieve major breakthroughs in process optimization, both for upstream and downstream processing. Continuous processing is the next step in process development which leads to cost reduction, higher productivity and better quality control; the integration of upstream and downstream processes is seen as a major challenge. In this review, we will focus on the state‐of‐the‐art of miniaturized techniques for process development in the biotechnology industry, and how automation and miniaturization drive process development. A comparison between liquid‐handling stations and microfluidics is made and an indication is given of which tools are still lacking for HTPD in the context of Integrated Continuous Biomanufacturing. © 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

White paper on high-throughput process development for integrated continuous biomanufacturing.

Continuous manufacturing is an indicator of a maturing industry, as can be seen by the example of the petrochemical industry. Patent expiry promotes a price competition between manufacturing companies, and more efficient and cheaper processes are needed to achieve lower production costs. Over the last decade, continuous biomanufacturing has had significant breakthroughs, with regulatory agencies encouraging the industry to implement this processing mode. Process development is resource and time consuming and, although it is increasingly becoming less expensive and faster through high‐throughput process development (HTPD) implementation, reliable HTPD technology for integrated and continuous biomanufacturing is still lacking and is considered to be an emerging field. Therefore, this paper aims to illustrate the major gaps in HTPD and to discuss the major needs and possible solutions to achieve an end‐to‐end Integrated Continuous Biomanufacturing, as discussed in the context of the 2019 Integrated Continuous Biomanufacturing conference. The current HTPD state‐of‐the‐art for several unit operations is discussed, as well as the emerging technologies which will expedite a shift to continuous biomanufacturing.

Design and development of a new ambr250\xae bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (NJS) and associated specific power input (P/V)JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

High throughput process development for the purification of rapeseed proteins napin and cruciferin by ion exchange chromatography

Proteins derived from plant resources such as oilseed meals, canola and sunflower, are considered a viable alternative to animal proteins for food consumption. This work presents a rational methodology, using high throughput experimentation (HTE), for the separation of cruciferin and napin, the two major proteins of canola meal, by chromatography. Eight different mixed mode and ion exchange resins were evaluated at different conditions with the aim of capturing napin and identifying adsorption/desorption behavior, ease of desorption and selectivity. POROS 50 HS resulted as the most promising resin. The obtained equilibrium adsorption data for napin and cruciferin was used in a mechanistic chromatography model and compared with experimental results showing a very good agreement. The model was used to identify column operating parameters that lead to >98% yield and purity for both proteins. Subsequently a conceptual downstream processing was proposed.

High-Throughput Process Development: I-Process Chromatography.

Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in the removal of product-related, host-cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during the development of a chromatographic step. Traditional process development is performed as a trial-and-error-based evaluation and often leads to a suboptimal process. A high-throughput process development (HTPD) platform involves the integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatographic process development. Creation of such platforms requires the integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing the HTPD of chromatography steps. It describes the operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6μL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.93). We think that this protocol will be of significant value to those involved in performing the high-throughput process development of the chromatography process.

Media Selection in Ion Exchange Chromatography in a Single Microplate.

High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select ion exchange chromatographic media using a 96-well filter microplate.

High-Throughput Process Development: II-Membrane Chromatography.

Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter, including high-pressure drop across the column bed and dependence on intraparticle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular, monoclonal antibody products. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing HTPD of a membrane chromatography step. It describes the operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with a 7μL membrane in each well. We will discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion-exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.9866). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography.

High-Throughput Process Development for the Chromatographic Purification of Viral Antigens.

Chromatography is a widely used method in the biotechnology industry and functions to separate the desired product from process and product related impurities. There is a multitude of resins available based on different modalities (such as charge, hydrophobicity, and affinity) to provide a spectrum of approaches to meet the separation challenges of the diverse products. The challenge of developing viral antigen purification processes is addressed in this method. A unique feature of this product class is that in order to protect against more than one strain of an antigen, vaccines are often multivalent. This entails multiple production processes for each antigen, all of which will require separate development and validation. Ideally, a universal purification method is sought, but differences in the protein subunits (frequently used as the antigens) make this challenging and often-bespoke purification steps are required. This means process development for the chromatographic stages of these products can be particularly challenging and labour intensive. With the numerous choices available, making critical process decisions that are usually unique to each product, process, and strain, can be costly and time-consuming. To address this, scale down purification at <1.0mL column volume and automation approaches are increasingly applied to increase throughput. In this work, a method is described wherein a Tecan Freedom EVO® automated liquid handler is deployed for the evaluation of different resin chemistries and buffer conditions to find a suitable purification strategy. This method allows for the rapid evaluation of the separation viral antigens where limited information on chromatography behavior is known at the early stages of process development. Here, we demonstrate the methodology firstly by explaining the automated purification script and secondly by applying the script for an efficient purification development for different serotypes of rotavirus antigens.