Microbial technologies for biotherapeutics production: Key tools for advanced biopharmaceutical process development and control.

Current trends in the biopharmaceutical market such as the diversification of therapies as well as the increasing time-to-market pressure will trigger the rethinking of bioprocess development and production approaches. Thereby, the importance of development time and manufacturing costs will increase, especially for microbial production. In the present review, we investigate three technological approaches which, to our opinion, will play a key role in the future of biopharmaceutical production. The first cornerstone of process development is the generation and effective utilization of platform knowledge. Building processes on well understood microbial and technological platforms allows to accelerate early-stage bioprocess development and to better condense this knowledge into multi-purpose technologies and applicable mathematical models. Second, the application of verified scale down systems and in silico models for process design and characterization will reduce the required number of large scale batches before dossier submission. Third, the broader availability of mathematical process models and the improvement of process analytical technologies will increase the applicability and acceptance of advanced control and process automation in the manufacturing scale. This will reduce process failure rates and subsequently cost of goods. Along these three aspects we give an overview of recently developed key tools and their potential integration into bioprocess development strategies.

A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells.

The integration of physiological knowledge into process control strategies is a cornerstone for the improvement of biopharmaceutical cell culture technologies. The present contribution investigates the applicability of specific productivity as a physiological control parameter in a cell culture process producing a monoclonal antibody (mAb) in CHO cells. In order to characterize cell physiology, the on-line oxygen uptake rate (OUR) was monitored and the time-resolved specific productivity was calculated as physiological parameters. This characterization enabled to identify the tight link between the deprivation of tyrosine and the decrease in cell respiration and in specific productivity. Subsequently, this link was used to control specific productivity by applying different feeding profiles. The maintenance of specific productivity at various levels enabled to identify a correlation between the rate of product formation and the relative abundance of high-mannose glycoforms. An increase in high mannose content was assumed to be the result of high specific productivity. Furthermore, the high mannose content as a function of cultivation pH and specific productivity was investigated in a design of experiment approach. This study demonstrated how physiological parameters could be used to understand interactions between process parameters, physiological parameters, and product quality attributes.

Combining mechanistic and data-driven approaches to gain process knowledge on the control of the metabolic shift to lactate uptake in a fed-batch CHO process.

A growing body of knowledge is available on the cellular regulation of overflow metabolism in mammalian hosts of recombinant protein production. However, to develop strategies to control the regulation of overflow metabolism in cell culture processes, the effect of process parameters on metabolism has to be well understood. In this study, we investigated the effect of pH and temperature shift timing on lactate metabolism in a fed-batch Chinese hamster ovary (CHO) process by using a Design of Experiments (DoE) approach. The metabolic switch to lactate consumption was controlled in a broad range by the proper timing of pH and temperature shifts. To extract process knowledge from the large experimental dataset, we proposed a novel methodological concept and demonstrated its usefulness with the analysis of lactate metabolism. Time-resolved metabolic flux analysis and PLS-R VIP were combined to assess the correlation of lactate metabolism and the activity of the major intracellular pathways. Whereas the switch to lactate uptake was mainly triggered by the decrease in the glycolytic flux, lactate uptake was correlated to TCA activity in the last days of the cultivation. These metabolic interactions were visualized on simple mechanistic plots to facilitate the interpretation of the results. Taken together, the combination of knowledge-based mechanistic modeling and data-driven multivariate analysis delivered valuable insights into the metabolic control of lactate production and has proven to be a powerful tool for the analysis of large metabolic datasets.

Impact of apoptosis on the on-line measured dielectric properties of CHO cells.

Apoptosis is a common type of cell death in biopharmaceutical cell culture processes which causes decrease in viable cell density and product yield. The progression of apoptosis has been reported to influence the dielectric properties of mammalian cells; however, the on-line detection of these effects has been rarely described. This study provides a comprehensive analysis of the on-line detectability of dielectric changes upon apoptosis induction in an industrial fed-batch process of CHO cells expressing a recombinant monoclonal antibody. Using capacitance signals, measured at 25 frequencies, the impact of apoptosis on the dielectric spectra was investigated in eight bioreactor cultivations in which various process conditions were combined with two different apoptosis induction strategies (camptothecin treatment and glucose starvation). To differentiate the apoptosis-related information from the cell concentration-associated variance in the multivariate capacitance datasets, principal component analysis (PCA) was used. A second principal component, explaining an explicit proportion (>20%) of the variance, was identified to be related to dielectric changes induced by apoptosis. Furthermore, the analysis of caspase-3 and -7 activation and DNA fragmentation showed that the detected dielectric change occurred in the early phase of apoptosis. The presented results verify that apoptosis has a considerable impact on the dielectric features of CHO cells and it can be monitored on-line with the introduced tool-set combining capacitance measurement with multivariate data analysis.

Advanced Development Strategies for Biopharmaceutical Cell Culture Processes.

The shift from empirical to science-based process development is considered to be a key factor to increase bioprocess performance and to reduce time to market for biopharmaceutical products in the near future. In the last decade, expanding knowledge in systems biology and bioprocess technology has delivered the foundation of the scientific understanding of relationships between process input parameters and process output features. Based on this knowledge, advanced process development approaches can be applied to maximize process performance and to generate process understanding. This review focuses on tools which enable the integration of physiological knowledge into cell culture process development. As a structured approach, the availability and the proposed benefit of the application of these tools are discussed for the subsequent stages of process development. The ultimate aim is to deliver a comprehensive overview of the current role of physiological understanding during cell culture process development from clone selection to the scale-up of advanced control strategies for ensuring process robustness.

Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris.

Pichia pastoris is widely used for the production of recombinant proteins in industrial biotechnology. In general, industrial production processes describe fed-batch processes based on the specific growth rate. Recently, we introduced the specific substrate uptake rate (q s) as a novel parameter to design fed-batch strategies for P. pastoris. We showed that a dynamic feeding strategy where the feed was adjusted in steps to the maximum specific substrate uptake rate was superior to more traditional strategies in terms of specific productivity. In the present study, we compare three different dynamic feeding strategies based on q s for a recombinant P. pastoris strain with respect to cell physiology, methanol accumulation, productivity and product quality. By comparing (A) a feeding profile at constant high q s, (B) a periodically adjusted feeding profile for a stepwise q s ramp, and (C) a feeding profile at linear increasing q s, we evaluated potential effects of the mode of feeding. Although a dynamic feeding strategy with stepwise increases of q s to q s max resulted in the highest specific productivity, a feeding profile where the feeding rate was stepwise increased to a constant high q s value was superior in terms of the amount of active enzyme produced and in the amount of accumulated methanol. Furthermore, this feeding strategy could be run automatically by integrating an online calculator tool, thus rendering manual interventions by the operator unnecessary.

Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

Risk-based Process Development of Biosimilars as Part of the Quality by Design Paradigm.

In the last few years, several quality by design (QbD) studies demonstrated the benefit of systematic approaches for biopharmaceutical development. However, only very few studies identified biosimilars as a special case of product development. The targeted quality profile of biosimilars is strictly defined by the originator's product characteristic. Moreover, the major source of prior knowledge is the experience with the originator product itself. Processing this information in biosimilar development has a major effect on risk management and process development strategies. The main objective of this contribution is to demonstrate how risk management can facilitate the implementation of QbD in early-stage product development with special emphasis on fitting the reported approaches to biosimilars. Risk assessments were highlighted as important tools to integrate prior knowledge in biosimilar development. The risk assessment process as suggested by the International Conference on Harmonization (ICH Q9) was reviewed and three elements were identified to play a key role in targeted risk assessment approaches: proper understanding of target linkage, risk assessment tool compliance, and criticality threshold value. Adjusting these steps to biosimilar applications helped to address some unique challenges of these products such as a strictly defined quality profile or a lack of process knowledge. This contribution demonstrates the need for tailored risk management approaches for the risk-based development of biosimilars and provides novel tools for the integration of additional knowledge available for these products. LAY ABSTRACT: The pharmaceutical industry is facing challenges such as profit loss and price competition. Companies are forced to rationalize business models and to cut costs in development as well as manufacturing. These trends recently hinder the implementation of any concepts that do not offer certain financial benefit or promise a long return of investment. Quality by design (QbD) is a concept that is currently struggling for more acceptance from the side of the pharmaceutical industry. To achieve this, the major goals of QbD have to be revisited and QbD tools have to be subsequently developed. This contribution offers an example as to how implement risk management in early-stage biosimilar development as part of the QbD concept. The main goal was to go beyond the conventional QbD workflow and to adjust risk management to the challenges of biosimilar products. Accordingly, instead of using methods like failure mode and effects analysis, recommendations of the ICH Q9 guideline were reviewed and put into practice by creating tailored risk assessment tools. The novelty of this contribution is to report those tailored tools ready-to-use for early bioprocess development of biosimilars along QbD principles.

A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding.

Mixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time-consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly-control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs-controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical-specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time-dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment.