A multi-bioreactor system for optimal production of malaria vaccines with Pichia pastoris

The successful development of optimal multistage production processes for recombinant products with Pichia pastoris needs to meet three pre-conditions. These pre-conditions are (i) strategies for performing fully automated and observable processes, (ii) characterization of the host cell-specific reaction parameters in order to make an adapted process layout for feeding and aeration strategies, and (iii) knowledge of optimal operation parameter conditions for maximizing the expression productivity of target protein amount and/or quality. In this report, an approach of a fully automated multi-bioreactor plant is described that meets all these requirements. The expression and secretion of a potential malaria vaccine with Pichia pastoris was chosen as an example to demonstrate the quality of the bioreactor system. Methods for the simultaneous identification of reaction kinetics were developed for strain characterization. Process optimization was carried out by applying a sequential/parallel Design of Experiments. In the view of Process Analytical Technology (PAT)-applications and in order to develop fully automated and globally observable production processes, methods for quasi on-line monitoring of recombinant protein secretion titers and the immunological quality of the products are also discussed in detail.     

Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening

Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed‐batch processes, including at‐line sampling, deep freezer off‐line sample storage, and complete data handling. To enable scalable high‐throughput early‐stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom‐built hardware and software, fully automated milliliter‐scale microbial fed‐batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high‐throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early‐stage bioprocess development.     

Process optimization using combinatorial design principles: parallel synthesis and design of experiment methods

The use of parallel synthesis techniques with statistical design of experiment (DoE) methods is a powerful combination for the optimization of chemical processes. Advances in parallel synthesis equipment and easy to use software for statistical DoE have fueled a growing acceptance of these techniques in the pharmaceutical industry. As drug candidate structures become more complex at the same time that development timelines are compressed, these enabling technologies promise to become more important in the future.     

Traces matter: Targeted optimization of monoclonal antibody N-glycosylation based on/by implementing automated high-throughput trace element screening

The use of high-throughput systems in cell culture process optimization offers various opportunities in biopharmaceutical process development. Here we describe the potential for acceleration and enhancement of product quality optimization and de novo bioprocess design regarding monoclonal antibody N-glycosylation by using an iterative statistical Design of Experiments (DoE) strategy based on our automated microtiter plate-based system for suspension cell culture. In our example, the combination of an initial screening of trace metal building blocks with a comprehensive DoE-based screening of 13 different trace elemental ions at three concentration levels in one run revealed most effective levers for N-glycan processing and biomass formation. Obtained results served to evaluate optimal concentration ranges and the right supplementation timing of relevant trace elements at shake flask and 2 L bioreactor scale. This setup identified manganese, copper, zinc, and iron as major factors. Manganese and copper acted as inverse key players in N-glycosylation, showing a positive effect of manganese and a negative effect of copper on glycan maturation in a zinc-dependent manner. Zinc and iron similarly improved cell growth and biomass formation. These findings allowed determining optimal concentration ranges for all four trace elements to establish control on desired product quality attributes regarding premature afucosylated and mature galactosylated glycan species. Our results demonstrates the power of combining robotics with DoE screening to enhance product quality optimization and to improve process understanding, thus, enabling targeted product quality control.     

Transfer of an adherent Vero cell culture method between two different rocking motion type bioreactors with respect to cell growth and metabolic rates

Rapid and simple cell and virus cultivation can currently be carried out using disposable bioreactors. The CELL-tainer^® (CELLution Biotech BV) disposable bioreactor is a rocking-type bioreactor which not only has vertical movement but horizontal displacement as well. Due to this two-directional movement relatively high mass-transfer capacities can be reached when compared with conventional rocking motion-type bioreactors.Using the design of experiments (DoE) approach we have developed models for the mixing times in both the CELL-tainer^® and the BIOSTAT^® CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type). The conditions for cultivation of Vero cells in the CELL-tainer^® bioreactor were chosen based on comparable mixing times.Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer^® and in the BIOSTAT^® CultiBag RM. Both bioreactors were controlled with regard to temperature, pH and % dissolved oxygen. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, polio virus production in both bioreactors was shown to be similar.     

A comparison of inoculation methods to simplify recombinant protein expression screening in Escherichia coli

In the past five years, Structural Genomics (SG) initiatives have established an automated pipeline for protein production in Escherichia coli to rapidly screen various conditions, resulting in soluble expression of recombinant proteins to aid in carrying out structural studies. However, some steps of the procedure are still extensive and require manual handling. Here, we present a comparative study of one step of the process, E. coli cultivation, using a set of 12 expression vectors encoding for fusion proteins of seven independent target proteins. First, we show that performing E. coli growth in auto-inducible medium (ZYM-5052) results in a comparable protein expression/solubility profile to that obtained when growing cells in classical Luria-Bertani (LB) medium. Second, we show that the transformation mix can be used directly to inoculate a culture, saving time and circumventing the error-prone step of colony picking, without impairing cell growth and the protein expression/solubility profile. Thus, we show that a basic, but nevertheless essential, step of a protein production pipeline, E. coli cultivation, can be simplified to a single event that is fully compatible with complete automation.     

Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.     

Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.     

Studies on the maximization of recombinant Helicobacter pylori neutrophil-activating protein production in Escherichia coli: application of Taguchi robust design and response surface methodology for process optimization

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) is a major antigen responsible for the generation of immune response in an infected individual. The cloning and expression of the gene corresponding to neutrophil-activating protein (NAP) were followed by process development for enhanced production and purification. The production process was developed in two parts. In the first part, some of the cultivation medium components (viz. carbon to nitrogen ratio, concentrations of sodium polyphosphate and magnesium sulphate) were optimized using the Taguchi robust experimental design. The intracellular NAP production level after 24 h of cultivation was considered as the target function or the dependent variable. There was a 76.8% increase in the NAP production level. Using this optimal medium composition obtained in the first part, the temperature of cultivation and the pH of cultivation medium were optimized in the second part. The NAP production level at hour 30 of cultivation was considered as the target function or the dependent variable. The optimal values for these two independent variables were 37.2 °C and 6.3 respectively. At this combination of temperature and pH, the theoretical maximum NAP production level was 1280 mg l^–1. This optimal combination was verified experimentally and the NAP production level was found to be 1261 mg l^–1. The optimization of the cultivation conditions resulted in a 61.5% increase in NAP production level. About a 2.91-fold overall increase in NAP production level at hour 24 of cultivation was achieved through process optimization.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads

Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek^® Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.     

An integrated scale-down plant for optimal recombinant enzyme production by Pichia pastoris

An integrated bioprocess was created in a scale-down production plant by developing a two-stage enzyme production process with Pichia pastoris, containing a cell-breeding reactor and a production reactor in combination with a three-stage downstream process. To harvest the secreted enzymes, a disc separator and a cross-flow microfiltration clear the broth from the cells. Purification with hydrophobic interaction chromatography removes other proteins, concentrates the product, and prepares the enzyme solution for lyophilization. Fully automated and broad observable multi-stage parallel process courses have been developed using industrial process control systems and at-line measurements for enzyme concentration and enzyme activity. Optimal process conditions were found by application of Design of Experiments (DoE) for the production process.     

Model based optimization of the fed-batch production of a highly active transglutaminase variant in Escherichia coli

A process for the production of a thermostable variant of a microbial transglutaminase was developed. The transglutaminase variant produced, carried a single amino acid exchange (serine replaced by proline at position 2) and showed a nearly doubled specific activity of 46.1 Umg(-1) compared to the wild-type enzyme. Based on a model based optimization strategy, intracellular soluble production in Escherichia coli was optimized. After parameter identification and only two fed-batch cultivations, a space time yield of 1438 U(TG)L(-1)h(-1) was obtained which is 175% higher than the highest values published so far (extracellular production using Corynebacterium ammoniagenes). High carbon source concentrations during expression were found to increase the product formation. Prior to the fed-batch cultivation, the host strain was adapted from complex medium to minimal medium by serial dilution. Upon transfer to the minimal medium, initially the maximal growth rate dropped to 0.13 h(-1). After the six consecutive cultivations the rate increased to 0.47 h(-1) and the portion of the complex medium was reduced to 1 ppm. Using the adapted cells, temperature after induction and IPTG-concentration were investigated by satellite batch cultivation according to a Design of Experiment (DoE) plan. The product yield was strongly influenced by the temperature after induction but not by the inductor concentration. The highest specific activity of 1386 Ug(-1) bio dry mass was obtained at 29°C and 0.7 mM IPTG.     

Design of experiments reveals critical parameters for pilot‐scale freeze‐and‐thaw processing of L‐lactic dehydrogenase

Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze‐and‐thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L‐lactic dehydrogenase (LDH) in a 700‐mL pilot‐scale freeze container equipped with internal temperature and pH probes. In 24‐hour experiments, target temperature between –10 and –38°C most strongly affected LDH stability whereby enzyme activity was retained best at the highest temperature of –10°C. Cooling profile and liquid fill volume also had significant effects on LDH stability and affected the protein aggregation significantly. Parameters of the thawing phase had a comparably small effect on LDH stability. Experiments in which the standard sodium phosphate buffer was exchanged by Tris‐HCl and the non‐ionic surfactant Tween 80 was added to the protein solution showed that pH shift during freezing and protein surface exposure were the main factors responsible for LDH instability at the lower freeze temperatures. Collectively, evidence is presented that supports the use of DoE‐based systematic analysis at pilot scale in the identification of F/T process parameters critical for protein stability and in the development of suitable process control strategies.     

Split-enzyme fragment as a single affinity tag that enables protein expression,purification, and functional assays

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.     

Development and optimization of several stages of the technological process of filgrastim substance production

Technology of industrial production of an active pharmaceutical substance (APS) of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) involved the use of a highly productive E. coli strain capable of biosynthesis of rhG-CSF in the form of inclusion bodies (IB). A method of strain cultivation has been described, and methods of IB isolation, industrial-scale purification, filgrastim APS production, and quality control have been developed. Clinical trials of the preparation, carried out in the leading Russian clinics, were successful. Efficiency and safety of the preparation have been confirmed. A ready pharmaceutical form Neupomax® been produced in Russia since 2008 according to the technology developed.     

Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k(L)a

Fermentation optimization experiments are ideally performed at small scale to reduce time, cost and resource requirements. Currently microwell plates (MWPs) are under investigation for this purpose as the format is ideally suited to automated high-throughput experimentation. In order to translate an optimized small-scale fermentation process to laboratory and pilot scale stirred-tank reactors (STRs) it is necessary to characterize key engineering parameters at both scales given the differences in geometry and the mechanisms of aeration and agitation. In this study oxygen mass transfer coefficients are determined in three MWP formats and in 7.5 L and 75 L STRs. k(L)a values were determined in cell-free media using the dynamic gassing-out technique over a range of agitation conditions. Previously optimized culture conditions at the MWP scale were then scaled up to the larger STR scales on the basis of matched k(L)a values. The accurate reproduction of MWP (3 mL) E. coli BL21 (DE3) culture kinetics at the two larger scales was shown in terms of cell growth, protein expression, and substrate utilization for k(L)a values that provided effective mixing and gas-liquid distribution at each scale. This work suggests that k(L)a provides a useful initial scale-up criterion for MWP culture conditions which enabled a 15,000-fold scale translation in this particular case. This work complements our earlier studies on the application of DoE techniques to MWP fermentation optimization and in so doing provides a generic framework for the generation of large quantities of soluble protein in a rapid and cost-effective manner.