Confirmation that ReadyKleer™ bioreactor single-use bag film does not impact cell culture performance and has equivalency with existing film material

To increase security of supply we have introduced in-house manufactured single-use bags for Xcellerex™ XDR bioreactors, in addition to our existing, long-standing offering. These new bags are manufactured using our well-established ReadyKleer™ film. This study, conducted in triplicate, was instigated to verify cell culture performance comparability between the two film materials.

ReadyKleer™ film demonstrated no impact on cell culture performance when compared with the existing film and is fitting for use in the Xcellerex™ XDR bioreactor range.

The two bag films showed equivalency during cell harvesting for:

  • Viable cell density (VCD) / cell viability (Figures 3 and 4).
  • Titer (Figure 5).
  • Metabolites – glucose and lactate concentration (Figures 6 and 7).
  • Product quality – aggregate, main and fragment distribution / charge distribution (Figures 8 and 9).

Introduction

Having a robust supply chain with an emphasis on security of supply is key to mitigating risk in biomanufacturing.

One of our most established products, the Xcellerex™ XDR bioreactor, at the time of publishing, has over 2500 units installed worldwide. Ensuring the single-use bags that form part of these bioreactors are readily available, is vitally important to the timely and continuous production of biotherapeutics. Always seeking opportunity to reduce risk and ensure continuance of supply, we now manufacture single-use bags at our own centers of excellence, as an addition to the legacy bags manufactured by our long established CMO, Thermo Fisher Scientific. ReadyKleer™, the film chosen to manufacture these new bags, is well-established across the industry and already used in a number of our single-use products, such as the Xcellerex™ XDR-10. See Figures 1 and 2 for a breakdown of bag film material composition.

To provide confidence that these new bags do not impact cell culture performance, we have conducted a verification study in triplicate. Three runs were performed using Chinese hamster ovary (CHO) cell fed-batch cultivation on the Xcellerex™ XDR-50 bioreactor with the ReadyKleer™ film. The performance was compared against two runs performed using identical conditions but with the legacy bag film.

Comparison of cell growth, viability, product titer, and product quality parameters were used to determine equivalency between the two bag films.

Fig 1. Illustrated cross-section of ReadyKleer™ film (layers measure in mil).

 

Fig 2. Illustrated cross-section of legacy film showing inner and outer layers (measured in mil).

 

Materials and methods

Cell line and culture medium

CHO cell line expressing the immunotherapeutic mAb, Herceptin (Genentech Inc.), was used in all experiments. We used HyClone™ ActiPro™ cell culture medium supplemented with 6 mM L-glutamine.

Fed-batch process in Xcellerex™ XDR-50

Experiments were performed at Cytiva laboratories, USA in 2023.

The Xcellerex™ XDR-50 bioreactors were all inoculated at a cell density of 0.3 × 106 cells/mL in a total volume of 35 L. Feeding of the bioreactor took place between Day 3 and 13 with 3% and 0.3% HyClone™ Cell Boost™ 7a and Cell Boost™ 7b feed supplements respectively, to support the nutrient demands. Daily bolus feed to 5 g/L started at Day 5, or earlier if the glucose concentration fell below 2 g/L.

200 mM L-glutamine was fed to the bioreactor on Day 4 to ensure concentration did not fall below 6 mmol/L. On Days 5 and 6 a feed was performed to ensure concentration did not fall below 2 mmol/L. This glutamine feed strategy was only required for runs 2 and 3. During run 1 on Day 4, glutamine was fed to 6 mmol/L.

Sampling plan

Daily sampling of the reactors was performed, and samples were tested for viable cell density (VCD), percentage viability, metabolites, and average cell diameter as well as osmolarity.

Samples for product quality (charge variants, aggregate, and glycan profile) were taken at Day 7 and at harvest (Day 13).

Results

50 L runs were successfully harvested. Graphs highlighting comparability attributes such as viable cell density, cell viability, titer, metabolite profiles (glucose and lactate concentration), and product quality (aggregate, main and fragment distribution / charge distribution) are shown in Figures 3 – 9 below. Any variability between runs is typical of the level of variation seen for this cell line.

Fig 3. Viable cell density.

 

Fig 4. Cell viability.

 

Fig 5. Titer.

 

Fig 6. Glucose concentration.

 

Fig 7. Lactate concentration.

 

Fig 8. Aggregate, main and fragment distribution.

 

Fig 9. Charge variant distribution.

 

Conclusions

Three cell culture runs using Xcellerex™ XDR bioreactor bags manufactured with ReadyKleer™ film demonstrated no impact on culture performance when compared with two runs using the legacy film. Viability was maintained above 80% between Days 0 through 8, and cell density above 13 million cells/mL from Day 5 to Day 8, meeting the acceptance criteria. In addition, titer and product quality was consistent with historic 50 L runs using the long-established legacy bag film.

Introduction of ReadyKleer™ bag film improves supply chain robustness and security of supply.

This study:

  • Confirmed no quality implications, demonstrating that ReadyKleer™ film can be used in Xcellerex™ XDR bioreactors.
  • Provides confidence that the two single-use bag films are equivalent and do not impact cell culture performance.
  • Shows that material composition differences between bag films do not affect cell growth.

Whether you are developing a new process or wanting the assurance of an alternative bag, Xcellerex™ XDR bioreactor bags can now be supplied from two different manufacturing locations.

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