An NIR-based PAT approach for real-time control of loading in Protein A chromatography in continuous manufacturing of monoclonal antibodies

Control of column loading in Protein A chromatography is a crucial part of development of robust and flexible process platforms for continuous production of monoclonal antibody (mAb) products. In this paper, we propose a control system that uses near infrared spectroscopy (NIRS) flow cells to accomplish the above. Two applications have been demonstrated using a periodic counter-current continuous chromatography setup. The first application involves use of single NIR flow cell before the inlet of the loading column to measure the concentration of mAb in the harvested broth. Measurement was in real-time (every 3 s) and within ±0.05 mg/ml, significantly better than making UV-based concentration estimations. The second application involved use of an additional NIR flow cell at the outlet of the loading column to measure column breakthrough in real time. The concentration data was transferred to a Python-based monitoring and control algorithm layered over a Cadence BioSMB system. The program could successfully run a three-column periodic counter current method on the BioSMB whereas controlling loading to ensure optimal resin utilization in each loading cycle phase based on precharacterized dynamic binding capacity models, whereas maintaining periodic elutions. The system was tested with multiple perturbations in harvest concentration, modeled after deviations that could arise downstream of a perfusion cell culture system. The results show that the proposed control is a spectroscopy-based process analytical technology tool that facilitates real time monitoring and control of loading in process chromatography. It is adaptable to any continuous chromatography equipment and is very well suited for implementation in a continuous mAb production train.     

Gamma irradiating chromatography columns enables bioburden-free integrated continuous biomanufacturing

An important consideration for integrated continuous biomanufacturing is that the downstream chromatography steps integrated with the bioreactor should maintain a low bioburden state throughout the entire duration of the operation. One potential strategy to achieve this is to start bioburden-free and functionally close the chromatography system. While chromatography skids themselves can be rendered bioburden-free, limitations exist in applying these methods to chromatography columns. The small column sizes used in continuous multicolumn chromatography enable gamma irradiation of disposable columns to render them bioburden-free. However, this approach has not been widely implemented, likely because gamma irradiation can negatively impact resin performance. Here, several protective mobile-phase modifiers were screened and shown to help chromatography resins retain naïve-like performance. Gamma irradiated columns were then integrated into perfusion bioreactors for continuous capture. Successful integrated continuous capture downstream of perfusion bioreactors for greater than 40 days using protein A, custom affinity, and non-affinity capture resins for multiple biologic modalities is demonstrated in development and commercial settings. No indications of time-based performance decline or bioburden growth have been observed. This strategy enables bioburden-free integrated continuous biomanufacturing operations and could allow full process closure and decreased environmental control requirements for facilities; thus, permitting simultaneous multi-product operations in a ballroom arrangement.     

An approach to integrated antibody production: Coupling of fluidized bed cultivation and fluidized bed adsorption

Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.The help of H. Schmitz, A. Bader, J. Gätgens and M. Halfar during the experiments is gratefully acknowledged. This work was partially funded by the ministry of science and research of the Federal Republic of Germany within the project Stoffumwandlung mit Biokatalysatoren.     

扩张柱床吸附层析回收纯化灌流培养生产的单克隆抗体

用扩张柱床吸附层析技术,一步回收纯化连续灌流培养的单克隆抗体。用Streamline SP阳离子交换介质在固定床柱XK16/20上进行条件摸索,扩张床柱Streamline25和50分别用于小规模条件优化和中试规模放大。培养液中的低浓度单抗经此步处理,浓缩10倍以上,纯度提高5～7倍,回收率＞90%,制备周期比固定柱床层析缩短一半以上。 根据培养液中单抗浓度的不同,一次处理量为18～50L,纯化规模由实验室水平（400mg）扩大至中试水平（2g）,生产成本和工艺复杂性大为降低。应用扩张柱床吸附层析技术,建立单克隆抗体回收纯化工艺,具有经济、简便、高效实用和良好的可放大性。     

Integrated continuous biomanufacturing platform with ATF perfusion and one column chromatography operation for optimum resin utilization and productivity

A new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.     

Considerations on the flow configuration of membrane chromatography devices for the purification of human basic fibroblast growth factor from crude lysates

Membrane chromatography possesses numerous advantages such as operation at high flow rates, low back pressure, ease of handling and scale up, which make the membrane adsorber process a viable alternative to conventional packed column chromatography. A purification process for the isolation of human recombinant basic fibroblast growth factor (FGF‐2) based on membrane chromatography was investigated using devices with different flow configurations. In the first process, the FGF‐2 capture step was performed with an axial flow device, while the alternative method achieved direct capture of FGF‐2 from unclarified cell lysate with a tangential flow device. In both processes, FGF‐2 purities exceeded 82% and the purified cytokine displayed high biological activity. Binding capacity (BC) from fermentation broth of the axial flow device was 28 mg/mL. This was 50% higher than the BC obtained with the tangential flow device under particle‐free supernatant conditions (18 mg/mL) and 150% higher compared to the BC achieved with unclarified cell lysate (11 mg/mL). While membrane chromatography in tangential flow mode omits clarification and thus reduces the number of stages in the downstream process, it displays lower peak resolution and leads to a lower overall process yield.     

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co²⁺-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl₂ and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm² each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.     

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end‐to‐end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three‐column periodic counter‐current chromatography for capture of the product followed by a three‐column con‐current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end‐to‐end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998–1009, 2017     

Integrated continuous production of recombinant therapeutic proteins

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost‐effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high‐density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four‐column periodic counter‐current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high‐density perfusion CHO cell cultures were operated at a quasi‐steady state of 50–60 × 10⁶ cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed‐batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time‐based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch‐column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non‐value‐added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. Biotechnol. Bioeng. 2012; 109: 3018–3029. © 2012 Wiley Periodicals, Inc.     

Continuous integrated antibody precipitation with two-stage tangential flow microfiltration enables constant mass flow

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn⁺⁺ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre-existing high-molecular-weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter-current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow-through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.     

Design and control of integrated chromatography column sequences

To increase the productivity in biopharmaceutical production, a natural step is to introduce integrated continuous biomanufacturing which leads to fewer buffer and storage tanks, smaller sizes of integrated unit operations, and full automation of the operation. The main contribution of this work is to illustrate a methodology for design and control of a downstream process based on integrated column sequences. For small scale production, for example, pre‐clinical studies, integrated column sequences can be implemented on a single chromatography system. This makes for a very efficient drug development platform. The proposed methodology is composed of four steps and is governed by a set of tools, that is presented, that makes the transition from batch separations to a complete integrated separation sequence as easy as possible. This methodology, its associated tools and the physical implementation is presented and illustrated on a case study where the target protein is separated from impurities through an integrated four column sequence. This article shows that the design and control of an integrated column sequence was successfully implemented for a tertiary protein separation problem. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:923–930, 2017     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.     

A new large‐scale manufacturing platform for complex biopharmaceuticals

Complex biopharmaceuticals, such as recombinant blood coagulation factors, are addressing critical medical needs and represent a growing multibillion‐dollar market. For commercial manufacturing of such, sometimes inherently unstable, molecules it is important to minimize product residence time in non‐ideal milieu in order to obtain acceptable yields and consistently high product quality. Continuous perfusion cell culture allows minimization of residence time in the bioreactor, but also brings unique challenges in product recovery, which requires innovative solutions. In order to maximize yield, process efficiency, facility and equipment utilization, we have developed, scaled‐up and successfully implemented a new integrated manufacturing platform in commercial scale. This platform consists of a (semi‐)continuous cell separation process based on a disposable flow path and integrated with the upstream perfusion operation, followed by membrane chromatography on large‐scale adsorber capsules in rapid cycling mode. Implementation of the platform at commercial scale for a new product candidate led to a yield improvement of 40% compared to the conventional process technology, while product quality has been shown to be more consistently high. Over 1,000,000 L of cell culture harvest have been processed with 100% success rate to date, demonstrating the robustness of the new platform process in GMP manufacturing. While membrane chromatography is well established for polishing in flow‐through mode, this is its first commercial‐scale application for bind/elute chromatography in the biopharmaceutical industry and demonstrates its potential in particular for manufacturing of potent, low‐dose biopharmaceuticals. Biotechnol. Bioeng. 2012; 109: 3049–3058. © 2012 Wiley Periodicals, Inc.     

连续流层析技术在亲和层析中的应用及生产放大评估

生物制药行业迅速发展,尤其是上游表达量的增加和规模的扩大,促使上游培养采用连续灌流方式,同时也推动了下游纯化生产工艺相应的采取连续纯化策略。以灌流培养的Fc融合蛋白为例,采用 BioSMB PD设备,对比了下游工艺亲和层析捕获步骤中单柱批次纯化和连续流层析纯化的样品纯度和收率,并在此基础上进行小试工艺放大和生产实际用量成本计算评估。连续流层析实现了上游灌流培养与下游亲和层析连续化的可行性,工艺稳定,回收率与批次纯化接近,但相比批次纯化,生产效率明显提高,填料载量提高,同时填料使用效率提高,生产成本显著降低。     

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.