Monoclonal antibody production in hollow-fiber bioreactors: Process control and validation strategies for manufacturing industry

Monoclonal antibody production in hollow-fiber bioreactors: Process control and validation strategies for manufacturing industry A. Handa-Corrigan,* S. Nikolay, C . Ferguson+

* D. Fletcher,+ S. Mistry,+ A. Young+ and

*School of Biological Sciences, University of Surrey, Guildford, United Kingdom ‘Murex Diagnostics Ltd., Temple Hill, Dar-ford, Kent, United Kingdom

This article describes an optimization strategy, with supporting data, that demonstrates that hollow-fiber bioreactors can be used reproducibly for the commercial manufacture of monoclonal antibodies (MCAs). Results are presented here that show that the duration of a production run can be precisely defined, based on celhlar metabolic activity and MCA productivity. Data are presented for MCA productivity in 5 and 10% calf serum, supplemented media, and in a serum-free medium. Considerations for the selection of production media are discussed with respect to MCA productivity, media costs, and downstream purification. The reproducible manufacture of MCAs in hollow-fiber bioreactors enables the necessary validation and quality control procedures to be implemented by the manufacturing industry.

Keywords: Optimization; validation; hollow-fiber bioreactor; reproducibility; monoclonal antibodies

Introduction

Hollow-fiber bioreactors are increasingly being used for the production of secreted products (e.g., monoclonal antibodies; MCA) from mammalian cells. ‘J The commercial manufacture of products in continuously perfused bioreactors has been met with considerable resistance because:

1.

2.

3.

4.

5.

The process control, optimization, and predictability of perfusion bioreactors are poorly understood; The scale-up potential of most perfusion technologies is limited; It is difficult to determine the time at which a production run should be terminated; The risks of contamination may be increased with long- term operation; and There are, as yet, no clear definitions of what comprises a product “batch” when the cultivation is effected continuously over extended periods.

Address reprint requests to Dr. A. Handa-Cotigan, School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, United Kingdom Received 15 December 1993; accepted 8 July 1994

In previous studies, we have shown that MCA production in hollow-fiber bioreactors can be carefully controlled and predicted, provided that routine monitoring of the physical and metabolic state of the cultivation process is implemented. This strategy has also been shown to be successful for stirred-tank bioreactor perfusion systems.4 The use of defined control strategies for perfusion cultures has not been widely implemented in industry. The majority of reports in the published literature show that although some measure- ments of the metabolic state of the culture are made, these are not incorporated into a defined optimization and control strategy for extended cultivation. For example, the method that is generally used for MCA production in perfused bioreactors involves the recirculation of a batch of medium until the glucose concentration has fallen below a certain value (e.g., 0.2 g 1-l). A fresh batch of medium is recir- culated through the system each time the glucose is de- pleted. ’ J In other cases, medium perfusion is increased periodically to maintain high cell density, viability, or productivity.6P7 For commercial manufacturing processes, validation and quality control are key considerations. When the metabolic and physical status of a long-term cultivation cannot be controlled, then reproducibility and predictability cannot be guaranteed. In this work we provide the necessary methodologies and performance data that demonstrate that

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MCA production in hollow-fiber bioreactors can be successfully validated and quality controlled for commercial manufacture. The hollow-fiber bioreactor selected for these studies was the Acusyst R or Celltronics system (Endotron- its , Minneapolis, Minnesota).

Materials and methods

Cell lines and cultivation conditions

The following mouse X mouse hybridoma cell lines were used in this study: AFP-27 (an IgG secretor) was cultivated in RPM1 1640

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supplemented with 5% new born calf serum (NBCS). WD-01 (an IgG secretor) was cultivated in RPM1 1640 supplemented with either 5 or 10% fetal calf serum (FCS). WD-02 (an IgG secretor) was cultivated in a defined serum-free medium, UltraCulture (Bio- Whittaker, Walkersville, Maryland). WD-08 (an IgM secretor) was cultivated in RPM1 1640 supplemented with 10% FCS.

Cells were maintained and expanded in the above media in a 5% CO, in an air incubator at 37°C. In the hollow-fiber bioreactor, the intracapillary space (IC) was perfused with RPM1 1640 basal medium supplemented with 450 mg dl- ’ glucose and 5 mM glutamine. The extra-capillary (EC) space was perfused with the same media formulations, as described above for each cell line.

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w Data from 717 - 814 hr post inoculation

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Figure 1 Monoclonal antibody and metabolic data profiles for an AFP-27 hybridoma cell line cultivated in a Celltronics hollow-fiber bioreactor

226 Enzyme Microb. Technol., 1995, vol. 17, March

Process control for monoclonal antibodies in HF-bioreactors: A. Handa-Corrigan et al.

Hollow-fiber bioreactor operational and optimization procedures

Monoclonal antibody production was carried out in Celltronics hollow-fiber bioreactors (New Brunswick Scientific, Edison, New Jersey). A total of 2 X 10’ viable cells were inoculated into the EC space. Full operating instructions and conditions have been described previously. 3 Optimization and control of MCA production was effected by changing the rates at which media were supplied and/or product and waste removed during the course of the cultivation. Routine biochemical and product assays were carried out to verify the metabolic state of the culture. MCA, glucose, glutamine, ammonia, and lactate concentrations were determined three times per week, as described previously.3 The metabolite and assay results were used to effect changes to one or more of the following pump rates:

tor can be attributed to the various reasons stated previously (e.g., poor mass transfer, cell loss). A further possible cause for loss of predictable MCA productivity could be attributed to the occurrence of a nonsecretory population of cells arising within the culture that can compete more readily for metabolites than the secretory population. This would result in increased loss of MCA productivity with time as the ratio between secretory and nonsecretory cells increased. lo It is our recommendation that the run length for commercial MCA production in the Celltronics bioreactor should be strictly defined within the period where MCA productivity is linearly proportional to cellular metabolic activity.

Reproducibility of production

1. When glucose or glutamine concentrations fell below 150 mg dl~ ’ and 2 mM. respectively, the basal medium addition rate was increased.

2. When lactate and ammonia concentrations were above 150 mg dl- ’ and 2.5 mM, respectively, basal medium addition, waste removal, and medium reversal rates were all increased.

3. When the MCA concentration in the harvest stream had increased, the supply of EC perfusion medium was also increased.

MCA and metabolite uptake or production rates were calcu- lated from relationships described previously.3

Commercial production requires that each production run for a given cell line can be repeated reproducibly. Figures 2 and 3 show the MCA productivity profiles for two hybridoma cell lines: AFP-27 and WD08, producing IgG and IgM MCAs, respectively. For both cell lines, linear relationships between MCA production and metabolic rates were observed (data not included). For both these and other cell lines tested in our laboratory, reproducible MCA productivity was obtained in repeated hollow-fiber cultivations. These results show that reproducible manufacture of MCAs can be achieved in the Celltronics hollow-fiber bioreactor provided that the optimization and control strategy recommended in this article is followed.

Results and discussion

Production run duration Medium optimization

Mammalian cells can be cultivated in hollow-fiber bioreactors for long periods of time (i.e., months).* Depending on the surface area available for cell growth and the hollow- fiber system configuration, extended cultivation times can result in poor mass transfer characteristics, poor cell growth, and viability and loss of cellular material out of the hollow-fiber unit.’ In the Celltronics hollow-fiber bioreactor, we demonstrated that production reproducibility and process control becomes unreliable for all cell lines after a period of 30 to 35 days postinoculation. Figure la shows a graph of MCA productivity versus time, during an 814-h production run. The results show that the MCA productivity increased to a maximum of 4.84 mg h - ‘, at 7 17 h. After this time period, the MCA productivity started to decline. This was associated with a significant cell loss (approximately lo6 cells ml - ‘) in the product harvest stream out of the bioreactor. Figure lb-e shows graphs of the MCA production rates versus the glucose-glutamine uptake rates and lactate-ammonia production rates for the above run, respectively. The results show that MCA production rates were linearly proportional to the uptake rates of glucose and glutamine up to 7 17 h. In addition, the MCA production rates were also proportional to the production rates of lactate and ammonia up to 7 17 h. However, after 717 h these linear relationships were not apparent.

Monoclonal antibody production in the Celltronics bioreactor can be carried out both in serum-supplemented and in serum-free media. The monthly requirements of these sup- plements are low (0.2-5 1) compared to other bioreactor systems. Table 1 compares the performances of two 28-day production runs, carried out in 5 and 10% FCS supplemented media, respectively. The results show that there was approximately 22% greater MCA produced in the 10% FCS -supplemented run, compared to the 5% FCS-supplemented

MCA Productton Rate mg/hr

Time (Hours)

+ Production Run 1 - Production Run 2

The loss of predictable MCA productivity after 1 Figure 2 Monoclonal antibody productivity for two production

month’s cultivation in the Celltronics hollow-fiber bioreac- runs of the AFP-27 hybridoma cell line

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1 4. Cumulative IgM produced (mg) /

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Figure 3 Monoclonal antibody productivity for two production runs of the WD-08 hybridoma cell line

cultivation. However, twice the amount of serum was used in the 10% FCS-supplemented run. Therefore, with respect to MCA productivity, the 10% FCS-supplemented medium is the obvious choice for production purposes. However, any medium selection procedure must also take into consideration two additional factors:

Serum costs: The use of 10% FCS -supplemented medium, as opposed to 5%, increased the monthly production costs by approximately f50. For commercial production, this relatively small sum was not a major consideration. With respect to total MCA productivity, the use of a 10% supplemented medium was commercially more beneficial. Downstream purification: The removal of contaminating serum proteins during MCA purification is a primary consideration. The use of high serum concentrations sig- nificantly raises purification costs due to increased pro- cessing requirements. In the hollow-fiber bioreactor runs presented above, the MCA was purified successfully from both the 5 and the 10% serum-supplemented media

because the ratio of MCA to contaminating serum mol- ecules was high in both of these media formulations.

Increases in MCA productivity associated with high serum-supplemented media use must be carefully assessed against further increases in downstream purification costs. The use of serum-free media for MCA production in hollow-fiber bioreactors is an alternative option that is highly recommended. High MCA concentrations can be achieved in low-protein, serum-free media, facilitating downstream purification.

Figure 4a-e shows an example of the MCA and metabolite rate profiles for an IgG secreting cell line (WD-02) cultivated in a serum-free medium (UltraCulture; BioWhit- taker). As with previous serum-supplemented runs, linear relationships between MCA production rates and metabolite rates were demonstrated. For successful hollow-fiber cultivations using serum-free media the cell line must be fully adapted and must show stable secretion over extended cultivation periods.

Conclusions

Hollow-fiber bioreactors can be used for the commercial manufacture of MCAs. In this article we have demonstrated the use of a simple optimization strategy for reproducible and predictable production of MCAs in both serum- supplemented and serum-free media cultivations. It is recommended that the duration of a production run is defined as that period in which MCA productivity is linearly proportional to cellular metabolic activity. For validation purposes, hollow-fiber cultivations can be shown to be reproducible when the optimization strategies described in this article are implemented. The formulation of standard operating procedures (SOPS) becomes simplified because the process control, run duration, media conditions, and run reproducibility can all be accurately defined.

Monoclonal antibody productivity may vary consider- ably in different serum-supplemented media formulations.

Table 1 Hollow-fiber bioreactor production run comparisons for an IgG secreting hybridoma cell line cultivated in media supplemented with 5 and 10% fetal calf serum (FCS)

Hollow-fiber cultivations of WD-01 hybridoma

Performance of production run 5% FCS-supplemented medium 10% FCS-supplemented medium

Run length RPM1 1840 basal medium used FCS used Total MCA productivity MCA production rate versus

ammoni*lactate production rates

MCA production rate versus glucos*glutamine uptake rates

28 days 28 days 104.75 I 85 I

0.25 I 0.5 I 1.20 g 1.47 g

Linear relationships Linear relationships

Linear relationships Linear relationships

MCA, Monoclonal antibody


Process control for monoclonal antibodies in HF-bioreactors: A. Handa-Corrigan et al.

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Figure 4 Monoclonal antibody and metabolic data profiles for a WD-02 hybridoma cell line cultivated in serum-free media

For hollow-fiber cultivations, the choice of serum concen- Acknowledgment tration is mainly dictated by downstream purification costs. However, as demonstrated in this article, the use of high serum concentrations need not always be problematic. The use of low-protein, serum-free media is preferable. In our experience, UltraCulture (BioWhittaker) and HL-1 (Ven- trex) are two serum-free media formulations that can be routinely used for MCA production in hollow-fiber bioreactors. We have demonstrated that continuous perfusion technology can be successfully used for the industrial man-

The authors gratefully acknowledge the Teaching Company Directorate, UK, for their contributions toward this project.

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Monoclonal antibody production in hollow-fiber bioreactors: Process control and validation strategies for manufacturing industry