Dynamics of recombinant protein production by mammalian cells in immobilized perfusion culture

Dynamics of recombinant protein production by mammalian cells in immobilized perfusion culture Takeyuki Sugiura* and Makoto Kakuzaki† *Laboratory for Bone Research, Discovery Research Laboratories, Research and Development Division and †Biopharmaceuticals Development Center, Hoechst Marion Roussel Limited, Kawagoe, Saitama, Japan In spite of the generally stable nature of immobilized perfusion culture, its profile of target protein production frequently shows variations. This might be explained by the drift in the metabolism of cultured cells. To address this issue, we performed a set of four Opticell bioreactor cultures producing recombinant anticogulant protein PCGFX. All the cultures lasted 40 –50 days with the oxygen consumption rate (OCR) mostly around 10 mmol min21; nevertheless, glucose and lactate metabolism was fluctuated with a parallel fluctuation in the recombinant protein productivity (RPP). The mean productivity of recombinant PCGFX was determined to be about 1.0 mg day21 for all the cultures. The statistical analysis revealed a significant correlation between the lactate production rate (LPR) and RPP in two cultures. A significant correlation was further found between average OCR and RPP in another culture where OCR was exceptionally lowered under serum-free conditions. No parameter significantly correlated with RPP in the remaining one culture; thus, the overt drift of RPP resulted, at least partly, from that of the cell metabolic activity and the present data should be helpful to explore a strategy for maximizing productivity. © 1998 Elsevier Science Inc. Keywords: Recombinant protein production; cell metabolism; perfusion culture; Opticell; PCGFX

Introduction Animal cells used for the production of therapeutic1 or diagnostic2 products are cultured with a specified bioreactor in batch, fedbatch, or continuous fashion.3 In view of the relatively low productivity of animal cells, continuous culture, or more specifically, perfusion culture is preferable over other types of cultures.4 In batch mode, the culture state, as represented by the cell density or concentrations of nutrients and metabolites, is invariably changed. As a result, the productivity of the target protein is generally proportional to the cell number5 which is, in turn, related to GCR.6 In contrast, perfusion culture is capable of maintaining favorable cell physiology, leading to fairly stable cell densities and nutrients concentrations in the medium. Despite the well-controlled culture conditions, a certain degree of variations in the productivity of the target protein is somehow inevitable in perfusion

culture.7,8 From the literature and our own experiences as well, immobilized perfusion culture is likely to be entailed with this sort of fluctuation; nonetheless, to our knowledge, there have been few reports aimed at addressing this issue. We thus decided to investigate the relationship between selected metabolic indicators and RPP, which can account for the fluctuation of RPP. The production of an anticoagulant factor, recombinant PCGFX (protein C fused with gla domain of factor X),9 in an Opticell bioreactor was chosen as a model system because of our fruitful experiences with the production of recombinant protein C in this particular immobilized culture system.10,11 Here we describe an attempt to relate RPP to some specific metabolic variables and from the results discuss the strategy to improve the recombinant protein productivity in perfusion culture by removing causes of its fluctuation as far as possible.

Materials and methods Address reprint requests to Dr. T. Sugiura, Hoechst Marion Roussel Limited 3–2, Minamidai 1-chome, Kawagoe, Saitama 350-11, Japan Received 13 June 1997; revised 3 December 1997; accepted 8 December 1997

Enzyme and Microbial Technology 22:699 –704, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

Cell line The CHO8-1 cell line expressing PCGFX at a high level was established in a previously described manner.9 The cells were

0141-0229/98/$19.00 PII S0141-0229(98)00007-6

Papers maintained in 175 cm2 T-flask with MEMa2 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS, Gibco), 0.5 mm methotrexate (MTX, Sigma, St. Louis, MO), and 0.1 mg ml21 vitamin K3 (Sigma) at 37°C in a humidified 5% CO2 and 95% air atmosphere.

Opticell culture Cultures producing recombinant PCGFX were conducted using the Opticell 5200R bioreactor (Charles River Biotechnical Service, Wilmington, MA) according to the manufacture’s manual. Approximately 2 3 108 CHO8-1 cells were used as inoculum into Opticore S-51. ES medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 7.5 or 10% FCS, 2 mm glutamine (Nissui), 2 g l21 glucose, 0.5 mm MTX, and 2 g l21 sodium bicarbonate was used as a growth medium. In the production phase, the serum concentration was reduced to 2% (production medium). In one experiment, serum-free medium composed of ES medium plus a combination of 10 mg ml21 bovine transferrin (Binding Site Limited, Birmingham, U.K.) and 1 mg ml21 bovine pancreatic insulin (Sigma) was utilized. Throughout the duration of the culture, the medium volume in the culture vessel was maintained around 1 l. The set point of medium temperature and pH was 36.8°C and 7.2, respectively. To avoid an abrupt localized increase in pH and to minimize osmolarity variations, we did not utilize base solutions for pH adjustment. Rather, the pH of the feed medium was increased to a moderate alkaline range. Both the feed and harvest media were stored at 4°C in a refrigerator. The harvested medium reservoir was normally exchanged every two days. At the same time, samples were taken through a specific port. After centrifugation, the supernatants were stored at 220°C until further analysis.

Analytical methods We measured the concentration of PCGFX by virtue of the enzyme-linked immunosorbent assay (ELISA) system essentially as described for protein C quantitation,12 except that in the PCGFX ELISA, PC02 and PC05 monoclonals served as a primary and secondary antibody, respectively. Both monoclonal antibodies raised against protein C13 recognize the distinctive epitopes common to protein C and PCGFX. Glucose determination was done by using a Beckman glucose analyzer. Lactate was quantitated by the enzymatic assay kit commercially available from Behringer Mannheim (Tokyo, Japan). Average OCR was calculated from several data points that were acquired at 12-h intervals between each sample withdrawal. GCR, LPR, and RPP were calculated with the previous equations.14

Statistics The correlation between RPP, average OCR, GCR, and LPR was evaluated with a Cricket Graph III (Computer Associates International, Inc., NY). The significance of the correlation was analyzed by the Student’s t-test. P values less than 0.05 were considered significant.

Figure 1 The first run of the Opticell culture for producing recombinant PCGFX: OCR (2) and pH (•) (A). The perfusion rate is indicated at the top whereas the serum content in the medium is at the bottom. Arrowhead represents the time point when the perfusion was started: GCR (F) and LPR (E) (B). White bars denote the productivity of recombinant PCGFX

impossible to count the cell number directly; therefore, the bioreactor accomplishes the adequate management of cell physiology by continuously monitoring OCR and supplying sufficient amounts of oxygen. In the initial growth phase, the medium plus high concentrations of the serum was employed. In the case where the OCR value ceased to rise before reaching a plateau (around 10 mmol min21), we commenced the perfusion to further raise it with the same medium rather than the production medium. Otherwise, at an apparent maximum of OCR, the cultivation entered the production phase in which the perfusion was done with the production medium containing a decreased level of the serum while periodically harvesting the conditioned medium. Upon the noticeable fall of OCR, the serum was shot to the culture in an attempt to reactivate the cultured cells. The perfusion rate was changed arbitrarily to maintain both saturated OCR values and an appropriate pH range (around 7). Throughout the cultures, pH and OCR were automatically monitored by the respective sensors installed in the bioreactor. The concentrations of glucose, lactate, and recombinant PCGFX in the removed samples were estimated manually as described in Materials and methods.

Results In total, four cultures were carried out in the Opticell bioreactor for the production of recombinant PCGFX. The cultures were started with inoculation of recombinant CHO8-1 cells into the ceramic core assembled into the culture system. Due to its porous configuration, cells are immobilized onto the core so firmly that cells never detach readily even after their disintegration. This feature makes it 700

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Experiment 1 The purpose of the first experiment was to see if there is a correlation between RPP and some metabolic indexes in the Opticell culture where we intended to hold OCR levels at maximum during the production phase (Figure 1). The OCR value rose immediately after start of the culture. When OCR stopped increasing at the value in excess of 10 mmol

Dynamics of recombinant protein production: T. Sugiura and M. Kakuzaki

Figure 3 The third run of the Opticell culture: OCR and pH (A) and GCR, LPR, and RPP (B). The symbols are used as in Figure 1 Figure 2 The second run of the Opticell culture: OCR and pH (A) and GCR, LPR, and RPP (B). The symbols are used as in Figure 1

min21, feed and harvest was initiated with ES medium containing 2% serum. Throughout the production phase, OCR was largely kept around 10 mmol min21, ranging from 8 –14 mmol min21. The high OCR values were sustained until the end of the culture (880 h after initiation). The perfusion rate was 1.0 or 1.5 l day21 depending on the culture state. The pH was controlled above 6.8 except for the very last stage where it dropped to 6.5. The RPP determined by the specific ELISA varied between 0.5–2 mg day21 with an average value of 1.0 mg day21. The statistical analysis indicated that RPP was significantly correlated with LPR (p , 0.05).

Experiment 2 The second cultivation was performed in order to confirm the finding in the first experiment that RPP was correlated well with LPR. As Figure 2 illustrates, in this case OCR was not elevated during the first 120 h for unknown reasons. We began perfusion at 0.25 l day21 with the use of the growth medium while OCR still remained low, after which OCR was increased promptly. From the results of the first run, we assumed that the cells reach almost confluence when the OCR value of 10 mmol min21 is attained; therefore, approximately at 310 h, the medium was switched to the production medium. Although OCR and pH were mostly kept above 10 mmol min21 and 7.0, respectively, OCR occasionally showed a sharp fall, implying an inconstant nature of the culture. RPP fluctuated between 0.5–1.5 mg day21 during the production phase of the 120 h culture. On average, RPP of recombinant PCGFX was 1.1 mg day21 which was quite similar to the value obtained in the first cultivation. Unex-

pectedly, no parameter was found to correlate with RPP; however, GCR and LPR showed a good correlation to each other (p , 0.01).

Experiment 3 We found a significant correlation between RPP and LPR in the first culture but not in the second one. This disappearance of the correlation might have been due to the unusual behavior of the second culture, characterized by the occasional sudden decrease in OCR values. To test this hypothesis, the third run was carefully conducted to reproduce the results of the first run in stable conditions (Figure 3). Progressive growth of CHO8-1 cells, as shown by an exponential increase in OCR, was observed just after the onset of the culture. Perfusion was started at 1.0 l day21 after 190 h of the culture in conjunction with replacement of the medium with the production medium. Subsequently, the perfusion rate was altered between 1.0 –2.0 l day21 for the purpose of stabilizing OCR values. There was a tendency that higher perfusion rates augmented both glucose consumption and lactate production. When the OCR value was attenuated gradually in response to the reduction of the perfusion rate from 2.0 to 1.0 l day21 (at around 380 h), 10% serum was shot to resume activity of the cells. The OCR was eventually recovered to the original value by about 600 h of the culture. All these efforts realized the highly stable culture in terms of OCR in the third experiment. The cultivation was continued for about 1,150 h, yielding a mean RPP of 0.92 mg day21, again very close to the values in the previous experiments, demonstrating the high reproducibility of the Opticell culture regarding the recombinant protein productivity. As expected, the statistical analysis showed a significant correlation between LPR and RPP (p , 0.05), substantiating the above hypothesis. Enzyme Microb. Technol., 1998, vol. 22, June

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Figure 4 The fourth run of the Opticell culture: OCR and pH (A) and GCR, LPR, and RPP (B). The symbols are used as in Figure 1

Besides, GCR was correlated with both RPP (p , 0.05) and LPR (p , 0.01).

Experiment 4 We performed a final culture to test the effect of a serum-free medium on metabolic parameters during the production phase. In parallel, we attempted to achieve an economic culture by using a lower amount of serum totally with retention of the productivity. To this end, we also lowered the serum content of the growth medium from 10 to 7.5% at the beginning of the culture. In spite of the serum level reduction, OCR was raised in the essentially same manner as before until 100 h of the culture after which the value started to decline (Figure 4); therefore, perfusion was initiated at 0.24 l day21 with the growth medium so as to raise OCR up to 10 mmol min21. While the OCR value was stabilized at around 580 h, the serum in the medium was completely depleted and replaced with a combination of growth factors, insulin, and transferrin. Remarkably unlike CHOB3 cells, a high producer of recombinant protein C,10 the serum-free medium caused a sudden change in OCR from 10 to 4.5 mmol min21, indicating that the CHO8-1 cell line was more serum-dependent than its cognate, CHOB3.

The RPP reduction occurred in parallel with the decline of OCR; yet, no concomitant decrease in GCR or LPR was observed. Rather during this duration, both metabolic parameters were slightly increased. After about 2 weeks of the switch to the serum-free medium, the culture was replenished with 5% serum in order to implement the cellular activation; thereafter, the production medium was returned to the 2% FCS-containing medium. Through these manipulations, OCR was steadily restored until it exceeded 10 mmol min21. Despite the lower amount of serum used, the average RPP amounted to 1.3 mg day21. This is comparable with the values of the other cultures. The significant correlation existed between RPP and average OCR (p , 0.01) and between GCR and LPR (p , 0.01), but not between RPP and LPR. The outcomes of the cultures are summarized in Table 1. We collected all the batches of the harvested media from four different runs and purified recombinant PCGFX by the method specifically devised for the g-carboxylated molecule.9 Because of either relatively less RPP of CHO8-1 or sufficient oxygen supply by Opticell,11,15 a large fraction (over 70%) of the produced recombinant protein was modified correctly as assessed from the final yield of the purified protein (data not shown).

Discussion Although immobilized perfusion culture is stable in general as exemplified by well-controlled cell densities or OCR, the production rate of the target protein frequently undergoes certain degrees of variations. It is conceivable that this drift in the productivity is attributed to the corresponding change in the cell metabolism; hence, with the aim of exploring the strategy to fulfill the optimized conditions, we sought to establish the causal link between the recombinant protein productivity and certain metabolic variables in the Opticell immobilized perfusion culture. Note that in this study, the relationship of the productivity with the metabolic parameters was pursued under the oxygen supply-controlled conditions over extended periods in the high-density perfusion culture. We focused on average OCR, GCR, and LPR as representative metabolic parameters for the following reasons: 1. Glucose and oxygen are major constituents involved in the energy generation process; 2. Lactate is derived from glutamine in addition to glucose,16 implicating that lactate production would possibly delineate the cellular metabolic activity in a broader sense than glucose consumption (see below)

Table 1 Summary of Opticell cultures producing recombinant PCGFX

Experiment

Culture duration (h)

PCGFX productivity (mg day21)

Observed correlation

1 2 3 4

880 1,180 1,150 1,060

1.0 1.1 0.92 1.3

RPP/LPR GCR/LPR RPP/GCR, RPP/LPR, GCR/LPR RPP/OCR, GCR/LPR

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Dynamics of recombinant protein production: T. Sugiura and M. Kakuzaki 3. Realtime measurements of glucose and lactate concentrations17 and oxygen consumption rate18,19 have been realized, making it possible to perform continuous culture properly based on these parameters 4. We have been most interested in the carbohydrate metabolism of animal cells including baculovirus-infected insect cells.20,21 We observed a significant correlation between LPR and RPP in the first culture but failed to find it in the following culture. Assuming that the discrepancy had originated from the unstable profile of the second culture, we conducted the third cultivation to verify the result of the first experiment. As expected, a significant correlation was found between LPR and RPP in the third culture. The fourth experiment was attempted to reduce the cost of the cultivation by decreasing the serum level of both the growth and production medium. As a result, the serum-depleted medium was not successful in supporting the culture of the CHO8-1 cells well, leading to a considerable decrease in the OCR values. In the last experiment, it was OCR that correlated well with PRR. It is possible to interpret these results based on the metabolic pathway as follows. The pathway of the recombinant protein production is extremely complicated such that various substances could be involved in this process. In principal, both energy and precursor generation are required for the production of recombinant proteins, then it seems reasonable to hypothesize that the recombinant protein production is closely associated with the total cellular activity which should be mainly represented by ATP production.22 The ATP production is described by the following equation:23 APR 5 6OUR 1 LPRv(FL/C)

(1)

where APR is the volumetric ATP production rate (nmol l21 h21), OUR is the volumetric oxygen uptake rate (nmol l21 h21), LPRv is the volumetric lactate production rate, and FL/C is the ratio of lactic acid produced from the carbohydrate to the total lactic acid produced (range of 0 –1). Opticell is designed to regulate the oxygen consumption at high levels irrespective of other factors,15 resulting in the relatively constant value of OCR. Under the conditions where we can assume that F L/C and OCR are virtually constant during the culture, ATP production should be proportional to the lactate production according to the above equation, implying that there occurs a correlation between RPP and LPR. In this way, we can explain the linear relationship between RPP and LPR in the experiments 1 and 3 where OCR was stable mostly above 10 mmol min21. It should be pointed out that another correlation was observed between GCR and RPP in the experiment 3. This might be an indirect consequence through the simultaneous occurrence of the correlation between RPP and LPR and between GCR and LPR (Table 1). In the experiment 4, LPR was not correlated with RPP probably because the serum-free medium conditions were unable to sustain the stable culture, causing an unusual reduction in OCR; however, LPR was somehow kept relatively constant during the entire culture. In this situation, the correlation between OCR and RPP can be predicted by

Eq. (1). This was the case for experiment 4. Conversely, this result suggests that maintaining active oxygen metabolism of the cultured cells is a prerequisite for the maximized productivity of the recombinant protein. Meanwhile in the second culture, no metabolic variable was correlated to RPP. Considering the overall unstable nature of the culture, the result was not unexpected since it should be impossible to apply Eq. (1) to describe some linear relationship unless the stable culture was accompanied by the constant values of either OCR or LPR. Furthermore, we cannot exclude the possibility that some other metabolic factor(s) inherent to the instability might mask the underlying correlation. In any case from these results, we conclude that the fluctuation of RPP can be attributed to the metabolic drift based on Eq. (1); however, the relationship is unraveled only when the whole culture is stable enough to apply the equation. On the other hand, the average PCGFX productivity was reproducible among four cultures (Table 1). According to Eq. (1), the ATP production associated with RPP depends on LPR and OCR with the assumption of the constant F L/C as described above. In all the cultures, OCR was fluctuated around 10 mmol min21 and LPR around 2.0 g day21 during the production phase despite the various degrees of the drifts which hid the metabolic correlation in some cultures; thus, we speculate that the reproducibility of RPP can be accounted for by the overall consistency of cell metabolism brought by the Opticell bioreactor. At high cell densities, the high metabolic load can induce the alteration in the environment very fast.19 This change would contribute to the drift in the metabolic rate which ultimately fluctuates RPP as shown in this work; hence, the results herein help establish the scheme for improving the productivity in stable perfusion culture at least for a sort of cell lines such as CHO cells by eliminating some critical causes of less productivity. The realtime measurements of OCR ensures its high and stable values in high density culture. Given enough oxygen provision, vigorous production of lactate would result in high production rates of the target protein as suggested in the present study. We can employ glucose as a limiting factor in this context because it is the most dominant energy source for animal cells.24 Glucose could be depleted from the production medium. Instead, it would be fed through a separate port. In this setting, the carbohydrate must be added until the lactate concentration becomes no longer increased. Additionally, we are able to predetermine the critical lowest level of glucose based on its K m value for the production of the protein of interest.20,25 The online measurements of glucose and lactate concentrations along with adequate computer control would implement this strategy to assure the maximal capability of the cells to produce the valuable recombinant protein in the perfusion culture.

Acknowledgments We thank Drs. E. Konz, H. B. Maruyama, and S. Tanaka for their continuous support. Enzyme Microb. Technol., 1998, vol. 22, June

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