Biphasic cultivation strategy for optimized protein expression and product quality

Transcription

1 Biphasic cultivation strategy for optimized protein expression and product quality Christian Kaisermayer 1, Andreas Gili 2, Robert Weik 3 1 GE Healthcare Life Sciences, Björkgatan 30, Uppsala, Sweden. 2 Dept. of Biotechnology, Univ. of Natural Resources and Life Sciences, Muthg. 11, 1190 Vienna, Austria; 3 Polymun Scientific, Donaustr. 99, 3400 Klosterneuburg, Austria First presented at the 23 rd ESACT meeting, Lille, France June 23-26, 2013

2 Abstract In biphasic cultivations, the culture conditions are changed to allow maximum recombinant protein expression after accumulating biomass during an initial phase. The influence of specific culture parameters is cell line dependent. Additionally, the impact of culture parameters on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a CHO cell line expressing an Epo-Fc fusion protein was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and ph were shifted. A fractional factorial design was used to evaluate the influence of cultivation temperature and ph on cell growth as well as on recombinant protein production and aggregation. All three parameters were influenced by the cultivation temperature. Additionally, an interaction between ph and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37 C and ph 7.05, a parameter shift to low temperature and acidic ph resulted in a 2.5-fold increase in the final product concentration and a decrease in the aggregate fraction from 75% to less than 1%. 2

3 Materials and methods Parallel batch cultivation of a CHO cell line in stirred-tank reactors (Minifors, Infors AG, Switzerland) CHO DUKX-B11 cells expressing an Epo-Fc fusion protein Cultivation medium: DMEM/Ham s F12 supplemented with additional glucose, glutamine and soy peptone Temperature set points [ C]: 28.5; 30.0; 33.5; 37.0 and 38.5 ph range: 6.75; 6.90 and 7.05 Analytics: cell concentration, viability, selected metabolites and product concentration. To analyze Epo-Fc conformation, the recombinant protein was purified on protein A (MabSelect SuRe, GE Healthcare Life Sciences) and then analyzed by size exclusion chromatography (Superose 6, GE Healthcare Life Sciences) 3

4 Materials and methods A central composite design at three temperature and ph levels was used to evaluate the impact of these parameters on cell growth, recombinant protein production and aggregation of Epo-Fc. As a non linear response to temperature was expected, the design was extended by two additional cultures at 28.5 C and 38.5 C to investigate curvature in the response. A fractional factorial design at resolution V was used. The experimental variability was assessed by three independent centerpoint experiments. 4

5 The cultivation temperature had a pronounced effect on cell growth. While no decline of the growth rate was observed at 38.5 C, temperatures lower than 37 C resulted in slower cell growth and a substantially lower peak cell concentration (Fig. 1). However, cultivation at lower temperature extended the process time and led to a substantially increased viable cell integral. Fig 1. Cell growth (A) and peak product conc. (B) under different cultivation conditions (parameter shift after 48 h). Error bars show one standard deviation of triplicate centerpoint experiments 5

6 The cell-specific recombinant protein production was constant at about 9 pg/(c d) in the investigated temperature interval. Nonetheless, the larger viable cell integral at lower temperatures increased the final product concentration 2.5-fold compared with the initial standard conditions (37 C, ph 7.05), as shown in Figure 1. Fig 1. Cell growth (A) and peak product conc. (B) under different cultivation conditions (parameter shift after 48 h). Error bars show one standard deviation of triplicate centerpoint experiments 6

7 The modeled response shows a clear curvature and the maximum product concentration is predicted for a temperature of about 30 C (Fig 2). The cultivation ph did not have a significant influence on the titer. Because of the extended plateau phase, the volumetric productivity of the cultures was also higher at low temperatures. A maximum of about 14 mg/(l d ) was reached in the cultures run at 33.5 C (center-point). Fig 2. Prediction plots for titer (A) and peak cell concentration (B) vs. temperature. Confidence interval 95%. 7

8 Aggregation of Epo-Fc was strongly dependent on the cultivation ph and varied between 78 and 0.7% in the experiments. An acidic ph was clearly beneficial and resulted in low aggregate concentrations (Fig 3). A shift to low cultivation temperatures further reduced Epo-Fc aggregation, demonstrating a synergistic interaction between the two process parameters (Fig 3). Fig 3. Prediction plot for aggregate concentration vs. ph at 30 C cultivation temperature (A), confidence interval 95%. Interaction plot for aggregate concentration vs. ph and temp. (B). 8

9 The maximum monomer concentration is predicted for a parameter shift to a cultivation temperature below 31 C and a ph below 6.85 (Fig 4). Fig 4. Monomer concentration in % vs. cultivation temperature and ph. 9

10 The influence of the culture conditions on product quality is also illustrated in the chromatograms in Figure 5, comparing cultivation at 30 C and ph 6.75 with the initial standard conditions of 37 C and ph Under low temperature and low ph conditions, about 3% of the recombinant protein was detected as fragments. The monomer peak contained more than 96% of the recombinant protein and less than 1% was detected in aggregates. At higher temperature and neutral ph, a similar aggregate fraction of about 4% was observed. However, only about 20% of the total recombinant protein was detected as monomer, while more than 75% was found in aggregates. Fig 5. Size exclusion chromatograms after protein A capture showing different Epo-Fc conformation depending on cultivation conditions. (A), 30 C, ph 6.75, (B) 37 C, ph

11 Cultivation at hypothermic conditions is frequently performed to maximize harvest titers. This is usually achieved by an improved culture longevity. Additionally, an increase in cell-specific recombinant protein production has also been observed. However, the latter response was shown to be cell line-specific [2]. An overall reduction of cell metabolism at low temperature settings is probably the main reason behind the maintained cell viability and the extended process time. Additionally, it has also been shown that hypothermic conditions can have an anti-apoptotic effect on cells [2]. 11

12 In our study, a temperature decrease to 33.5 C or lower increased the final Epo-Fc concentration about 2.5-fold. A similar temperature dependent response of Epo production has also been shown in other experiments [3]. However, protein aggregation was not investigated during these studies. Epo contains four cysteins giving rise to two disulfide bonds. It is prone to form aggregates due to reduction and subsequent random re-oxidation of the thiol groups [1, 4]. Protein aggregation decreases the useful fraction of the product and is especially undesirable in the case of Fc fusion proteins, as aggregates and monomers co-purify during the initial capture step and are difficult to separate during the later downstream processing. 12

13 We observed a drastically reduced aggregate formation at acidic cultivation ph. Aggregation most probably occurs after secretion of Epo-Fc and thus is unrelated to the protein-folding process itself. However, it seems likely that the conditions in the culture supernatant affect the recombinant protein and cause aggregation. Although investigating a ph range of 7.0 to 7.7, it has been found that more alkaline conditions facilitate the oxidation of free thiols and consequently increase protein aggregation [4]. Our results show that a combination of low cultivation temperature and acidic ph resulted in a low concentration of protein aggregates. 13

14 Conclusion Hypothermic conditions (< 33.5 C) more than doubled culture longevity A biphasic cultivation strategy involving a shift to low temperature resulted in a 2.5-fold titer increase Interaction between temp. and ph for aggregation of Epo- Fc Parameter shift to 30 C and ph 6.75 reduced the aggregate concentration of Epo-Fc from 75% to less than 1% compared to cultivation at 37 C and ph

15 References [1] Way JC et al. Protein Eng Des Sel. 18, (2005). [2] Becerra S et al. Biochem Eng J. 60, 1-8 (2012). [3] Yoon S et al. Biotech Bioeng. 89, (2005). [4] Wang T et al. J Pharm Biomed Anal. 72, (2013). 15

16 Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. MabSelect SuRe and Superose are trademarks of GE Healthcare companies. Minifors is a trademark of Infors AG, Switzerland. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information General Electric Company All rights reserved. First published June 2013 GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden 16