Accelerate Process Development Research Articles

Miniaturized pH Holographic Sensors for the Monitoring of Lactobacillus casei Shirota Growth in a Microfluidic Chip.

Bioreactors have been used both to develop new, and to improve bioprocess yields for, biopharmaceutical products. However, efforts to miniaturize bioreactors, in order to save costs and accelerate process development times, have been limited by the lack of on-site monitoring capabilities available at such scales. In this study, small volume (3 nL) nonconsumptive holographic sensors were integrated into a glass-PDMS microfluidic chip to monitor via a blue-shift in the resultant holographic replay wavelength, the change in pH during microbial growth of Lactobacillus casei ( L. casei) Shirota. Within the optimal growth pH range of L. casei, the accuracy of the miniaturized pH sensors was comparable to that of a conventional pH meter. Conceivably, this approach could be extrapolated to an array of miniaturized holographic sensors sensitive to different analytes, and thereby paving the way for reliable, real-time, noninvasive monitoring of microorganisms in a nanobioreactor.

On-demand manufacturing of clinical-quality biopharmaceuticals.

Conventional manufacturing of protein biopharmaceuticals in centralized, large-scale single-product facilities is not well-suited to the agile production of drugs for small patient populations or individuals. Solutions for small-scale manufacturing are potentially more nimble, though previous systems are limited in both process reproducibility and product quality, owing to complicated means of protein expression and purification1–4. We describe an automated bench-top multi-product manufacturing system, called Integrated Scalable Cyto-Technology (InSCyT), for the end-to-end production of hundreds to thousands of doses of clinical-quality protein biologics in about three days. We also demonstrate that InSCyT can accelerate process development from sequence to purified drug in 12 weeks. We produced hGH, IFNα-2b, and G-CSF using highly similar processes on InSCyT and found that the purity and potency of these products is comparable to that of marketedreference products.

Optimization of Multiproduct Biorefinery Processes under Consideration of Biomass Supply Chain Management and Market Developments

Even though a shift from conventional to renewable resources is often envisaged, lignocellulosic biorefinery concepts struggle with economic viability and sustainability. In order to overcome these barriers, a full analysis from biomass supply chain, process performance optimization, and product-portfolio selection is targeted. Addressing the economic viability and sustainability already at an early process development stage when only limited knowledge is available, Process Network Flux Analysis (PNFA) [Ulonska et al., AIChE J. 2017, 62, 3096–3108] is capable of systematically identifying the most valuable processing pathways. This enables a first performance ranking based on the profit or global warming potential of pathways, thereby accelerating process development. While so far only processing networks have been considered, the methodology is herein extended to consider biomass supply chain optimization and market-dependent price developments such that all main influencing factors are considered simult...

Development of a general defined medium for Pichia pastoris.

Pichia pastoris is widely used as a host for recombinant protein production. More than 500 proteins have been expressed in the organism at a variety of cultivation scales, from small shake flasks to large bioreactors. Large-scale fermentation strategies typically employ chemically defined growth medium because of its greater batch-to-batch consistency and in many cases, lower costs compared to complex medium. For biopharmaceuticals, defined growth medium may also simplify downstream purification and regulatory documentation. Standard formulations of defined media for P. pastoris are minimal ones that lack the metabolic intermediates provided by complex components such as peptone and yeast extract. As a result, growth rates and per-cell productivities are significantly lower than in complex medium. We have designed a rich defined medium (RDM) for Pichia pastoris by systematically evaluating nutrients of increasing complexity and identifying those that are most critical for growth. We have also employed transcriptomics to gain deeper insights into the underlying metabolic processes and inform our media design. We have demonstrated that using RDM for expression of three heterologous proteins yields titers comparable to, or higher than, those in standard complex medium. RDM improves productivity of P. pastoris fermentations and its development demonstrates the usefulness of transcriptomics to accelerate process development for new molecules.

Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations.

Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of200-400U(gxh)-1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01-0.35 with smooth change of D at a rate of 0.003 h-2. EPG production was found to be comparable with a qp of 400 U (gx h)-1 at D = 0.27 h-1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.

Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza.

Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015–2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains.

An heuristic-based selection process for organic solvent nanofiltration membranes

Abstract Organic solvent nanofiltration has emerged as a versatile tool for process intensification in chemical production. Due to the complex interactions between membrane, solvent and solute, the transport mechanism has not yet been clarified and it is rather difficult to identify the best membrane for a given separation problem. Currently, this is solved by time-consuming and costly membrane screening experiments. To accelerate process development an heuristic for membrane selection is required. Therefore, the rejections of different substances with varying functional groups out of a material class of specialty chemicals were measured. The most typical membrane materials (Polydimethylsiloxane and Polyimide) and a wide range of solvents were used. From these experiments, parameters were identified which influence the membrane performance. A combination of these parameters in an heuristic allows for the identification of the most suitable membrane for a given separation problem.

Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.

High throughput development of a non protein A monoclonal antibody purification process using mini‐columns and bio‐layer interferometry

Downstream purification process development for therapeutic mAbs is a critical step that must be accomplished under compressed timelines. High‐throughput sorbent screening has gained much attention for accelerating process development, based on the rapid acquisition of a large dataset. This study describes the development of a non protein A purification process for a mAb expressed in a CHO cell supernatant. Capture and intermediate steps were screened on three mixed‐mode and one cation exchange chromatography sorbents using 200 μL mini‐columns compatible with automation. Capacity and yield were evaluated using biolayer interferometry and mAb purity, using SEC‐HPLC. A scale‐up of the capture and intermediate steps using 2.5 cm id and 0.5 cm id lab‐scale columns packed with MEP HyperCel™ mixed‐mode sorbent and CM Ceramic HyperD® F cation exchange sorbent, respectively, led to an overall mAb recovery yield of 90% and an mAb purity close to 97%. The high‐throughput workflow applied here, both in terms of screening and analytics, was a key point to accelerate process development and achieve optimal mAb product purity and yield at minimized time and cost.

Automated small‐scale protein purification and analysis for accelerated development of protein therapeutics

Small‐scale protein purification presents opportunities for accelerated process development of biotherapeutic molecules. Miniaturization of purification conditions reduces time and allows for parallel processing of samples, thus offering increased statistical significance and greater breadth of variables. The ability of the miniaturized platform to be predictive of larger scale purification schemes is of critical importance. The PerkinElmer JANUS BioTx Pro and Pro‐Plus workstations were developed as intuitive, flexible, and automated devices capable of performing parallel small‐scale analytical protein purification. Preprogrammed methods automate a variety of commercially available ion exchange and affinity chromatography solutions, including miniaturized chromatography columns, resin‐packed pipette tips, and resin‐filled microtiter vacuum filtration plates. Here, we present a comparison of microscale chromatography versus standard fast protein LC (FPLC) methods for process optimization. In this study, we evaluated the capabilities of the JANUS BioTx Pro‐Plus robotic platform for miniaturized chromatographic purification of proteins with the GE ӒKTA Express system. We were able to demonstrate predictive analysis similar to that of larger scale purification platforms, while offering advantages in speed and number of samples processed. This approach is predictive of scale‐up conditions, resulting in shorter biotherapeutic development cycles and less consumed material than traditional FPLC methods, thus reducing time‐to‐market from discovery to manufacturing.

Automated disposable small-scale bioreactor for high-throughput process development: implementation of the 24 bioreactor array

Aim: A disposable reactor system (250 ml scale) has now been developed into a fully automated 24 reactor array which includes process parameter monitoring and control, advanced feed strategies, automated event triggering, robotic arm liquid handling and sample storage. Results: Process and analytical comparability was shown for a range of Pichia, Escherichia coli and CHO cell-culture bioprocesses up to pilot and commercial scale. This established the system as a scale-down model for process development and characterization of large-scale industrial processes. Conclusion: This work demonstrated the ability of the automated system to accelerate process development by executing a single statistical design of experiments, with a wider range of parameters, up to 3–5-times faster than conventional approaches.

A framework for real-time glycosylation monitoring (RT-GM) in mammalian cell culture.

Glycosylation is a critical characteristic of biotherapeutics because of its central role in in vivo efficacy. Multiple factors including medium composition and process conditions impact protein glycosylation and characterizing cellular response to these changes is essential to understand the underlying relationships. Current practice typically involves glycosylation characterization at the end of a fed-batch culture, which in addition to being an aggregate of the process, reflects a bias towards the end of the culture where a majority of the product is made. In an attempt to rigorously characterize the entire time-course of a fed-batch culture, a real-time glycosylation monitoring (RT-GM) framework was developed. It involves using the micro sequential injection (μSI) system as a sample preparation platform coupled with an ultra-performance liquid chromatography (UPLC) system for real-time monitoring of the antibody glycan profile. Automated sampling and sample preparations were performed using the μSI system and this framework was used to study manganese (Mn)-induced glycosylation changes over the course of a fed-batch culture. As expected, Mn-supplemented cultures exhibited higher galactosylation levels compared to control while the fucosylation and mannosylation were consistent for both supplemented and control cultures. Overall, the approach presented in the study allows real time monitoring of glycosylation changes and this information can be rapidly translated into process control and/or process optimization decisions to accelerate process development.

LEED-ND and Livability Revisited

This study examines LEED-ND’s criteria for Neighborhood Pattern and Design (NPD). LEED-ND was developed as a system for rating new neighborhoods on the sustainability of their planning. However, it has increasingly been adopted by cities as a de facto measure of “livable” neighborhood design and used to accelerate development processes. We hypothesize that these criteria do not area is Temescal, a gentrifying neighborhood in Oakland, CA. livability very highly. Furthermore, residents consistently rated and ranked NPD characteristics quite differently than did LEED-ND, system. We propose that a single set of weighted, prescriptive desired amenities of different communities.

EuroSciCon meeting on bioprocess miniaturization: development and optimization 26 November 2013, London, UK

The impetus to accelerate process development in the biopharmaceutical industry is driven by reducing costs and time to market. Shortening development times is critical to the success of the industry [1]. The miniaturization of bioprocessing assists in the generation of quantitative data, fast and efficiently. This helps inform bioprocess design and speeds translation to the manufacturing scale. The meeting reviewed individual operations in the bioprocess workflow highlighting the state-of-the-art in technology and research and innovation. A brief overview of the themes covered is discussed below.

Implementation of QbD for the development of a vaccine candidate

This case study provides an example of how Quality by Design (QbD) principles were applied to accelerate process development to manufacture a vaccine candidate at commercial scale. By leveraging an existing manufacturing platform process, a risk assessment was used to differentiate process parameters that could be defined using a combination of scientific and historical manufacturing knowledge from those that merited additional process characterization by experimentation. Select parameters, and their interactions, were evaluated by a Design of Experiment (DoE) series. This systematic approach required less time and fewer resources and resulted in the definition of a reliable and robust manufacturing process that meets regulatory requirements.

Single‐step affinity purification of enzyme biotherapeutics: A platform methodology for accelerated process development

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT).

Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis

Cell-free protein synthesis (CFPS) platforms are now considered a powerful tool for synthesizing a variety of proteins at scales from pL to 100 L with accelerated process development pipelines. We previously reported the advancement of a novel yeast-based CFPS platform. Here, we studied factors that cause termination of yeast CFPS batch reactions. Specifically, we characterized the substrate and byproduct concentrations in batch, fed-batch, and semi-continuous reaction formats through high-performance liquid chromatography (HPLC) and chemical assays. We discovered that creatine phosphate, the secondary energy substrate, and nucleoside triphosphates were rapidly degraded during batch CFPS, causing a significant drop in the reaction's energy charge (E.C.) and eventual termination of protein synthesis. As a consequence of consuming creatine phosphate, inorganic phosphate accumulated as a toxic byproduct. Additionally, we measured amino acid concentrations and found that aspartic acid was rapidly consumed. By adopting a semi-continuous reaction format, where passive diffusion enables substrate replenishment and byproduct removal, we achieved over a 70% increase in active superfolder green fluorescent protein (sfGFP) as compared with the batch system. This study identifies targets for the future improvement of the batch yeast CFPS reaction. Moreover, it outlines a detailed, generalized method to characterize and improve other CFPS platforms.

Evaluation of the advanced micro-scale bioreactor (ambr™) as a highthroughput tool for cell culture process development

Bio-pharmaceutical industries face an increasing demand to accelerate process development and reduce costs. This challenge requires high throughput tools to replace the traditional combination of shake flasks and small-scale stirred tank bioreactors. A conventional and widely used process development tool is the stirred tank reactor (STR) ranging from approximately 1L to 10L in working volume. Physical culture parameters such as pH, temperature and pO2 can be easily controlled in such systems. However preparation and operation of these systems are time and resource consuming. The ambr™ system from TAP Biosystems has the capabilities for automated sampling, feed addition, and control for pH, dissolved oxygen, gassing, agitation, and temperature. Here, through the evaluation of parameters including cell growth, viability, metabolite concentration and production titer during a fed-batch process using CHO cells producing a recombinant mAb, we assessed the reproducibility of the ambr™ system for standard conditions compared to 2L stirred tank bioreactors and the effects of parameter ranging between both culture systems, namely feed rate and pH ranging.

Automated disposable small scale reactor for high throughput bioprocess development: A proof of concept study

The acceleration of bioprocess development for biologics and vaccines can be enabled by automated high throughput technologies. This will alleviate the significant resource burden from the multi-factorial statistical experimentation required for controlling product quality attributes of complex biologics. Recent technology advances have improved clone evaluation and screening, but have struggled to combine the scale down criteria required for both high cell density cell culture and microbial processes, with sufficient automation and disposable technologies to accelerate process development. This article describes the proof of concept evaluations of an automated disposable small scale reactor for high throughput upstream process development. Characterization studies established the small scale stirred tank disposable 250 mL reactor as similar to those of lab and pilot scale. The reactor generated equivalent process performance for industrial biologics processes for therapeutic protein and monoclonal antibody production using CHO cell culture, Pichia pastoris and E. coli. This included similar growth, cell viability, product titer, and product quality. The technology was shown to be robust across multiple runs and met the requirements for the ability to run high cell density processes (>400 g/L wet cell weight) with exponential feeds and sophisticated event triggered processes. Combining this reactor into an automated array of reactors will ultimately be part of a high throughput process development strategy. This will combine upstream, small scale purification with rapid analytics that will dramatically shorten timelines and costs of developing biological processes.

Microreactors with integrated UV/Vis spectroscopic detection for online process analysis under segmented flow

Combining reaction and detection in multiphase microfluidic flow is becoming increasingly important for accelerating process development in microreactors. We report the coupling of UV/Vis spectroscopy with microreactors for online process analysis under segmented flow conditions. Two integration schemes are presented: one uses a cross-type flow-through cell subsequent to a capillary microreactor for detection in the transmission mode; the other uses embedded waveguides on a microfluidic chip for detection in the evanescent wave field. Model experiments reveal the capabilities of the integrated systems in real-time concentration measurements and segmented flow characterization. The application of such integration for process analysis during gold nanoparticle synthesis is demonstrated, showing its great potential in process monitoring in microreactors operated under segmented flow.