Dénes Zalai

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.

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.

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.

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 originators 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 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.

A model system and chemometrics to develop near infrared spectroscopic monitoring for Chinese hamster ovary cell cultivations

Near infrared (NIR) spectroscopy is an ideal tool for biopharmaceutical process monitoring, as it can be used to generate information on key bioprocess variables rapidly online. Mammalian cell cultivation is a rapidly developing field of biopharmaceutical production where one of these key process variables is the glucose concentration. However, calibrations for the NIR-based monitoring of glucose are usually not available in the early phase of process development owing to the lack of sufficient NIR data from small-scale experiments. This article demonstrates the development of calibrations based on NIR spectroscopy (11,988.0–4297.0 cm−1) for the determination of glucose concentration in a novel shake flask model system for mammalian cell cultivation. To generate a homogeneous distribution of glucose concentration in the calibration range and to reduce the correlation between glucose and other metabolites, cultivation samples with low glucose levels were spiked with glucose, and NIR measurements were subsequently performed. Biochemical and physical variability was deliberately induced in the model system to mimic possible matrix effects typically occurring in fed-batch bioreactor cultivation samples. The specificity of the calibration model to glucose was increased by variable selection, so the spectral region between the two water peaks was used for calibration. Four mathematical pretreatments were evaluated by comparing the root-mean standard error of prediction (RMSEP) values of the models; the best preprocessing method was a novel combination of baseline offset method with the deresolve function. Our models predicted the glucose concentration in two test cultivations with an RMSEP of 3.12 mmol L−1 and 5.51 mmol L−1.

Investigation of heat-treated cultivation medium for mammalian cells with near infrared spectroscopy

A chemically defined medium powder for Chinese hamster ovary (CHO) cell cultivation was investigated regarding its response to heat treatments with different exposure times (1 hour, 7 hours and 13 hours) and temperatures (30 °C, 50 °C and 70 °C). The heat treatments were performed according to a design of experiments approach. Spectra of the control and the treated powders were collected to compare the sample groups using a dispersive and a Fourier transform (FT) near infrared (NIR) spectrometer. Multivariate data analysis including unsupervised classification as principal component analysis and supervised classification as soft independent modelling of class analogy was employed to identify the treatment-induced variations in the samples. Samples were separated according to the temperature set points of heat treatments and the control samples were successfully discriminated based on second-derivative NIR spectra. In order to evaluate the impact of the treatments on the growth rate of CHO cells, shake flask cultivation experiments were also performed. The viable cell density results showed that both treatment temperature and treatment duration were significant factors (p = 0.05). Additionally, the sensitivity of change detection for medium powders was higher in the case of NIR and FT-NIR measurement and spectrum analysis compared to traditional shake flask cultivation.