Single-use bioreactors (SUBs) are being increasingly utilized for biopharmaceutical manufacturing. Minimal requirements for cleaning, higher plant capacity due to quick turnaround and flexibility of manufacturing multiple products without extensive changeover are key attributes that have led to rapid adoption of disposable technology for cell culture manufacturing. Xcellerex XDR SUBs ranging from 50 L to 2000 L have been proven as successful replacement for stainless steel stirred-tank bioreactors. Xcellerex SUBs feature a cylindrical bag that uses a magnetically coupled, bottom-driven impeller with easy installation prior to culturing cells. The impeller is positioned at an angle, 15° off-centered at the bottom of the bag. Bioreactor bags with pre-installed disc spargers (micro and macrospargers) or open-pipe spragers can be used depending on the process. Sparge discs in Xcellerex SUB bags are fabricated on the impeller assembly. Stainless steel bioreactors are typically used with a top-mounted stirrer shaft installed with one or more impellers depending on the vessel size and process requirement. Stainless steel bioreactor tanks are also known to use baffles to prevent vortex while mixing the culture. Impellers in Xcellerex SUBs are positioned off-center which prevent vortex formation and thus does not require baffles for proper mixing. Stainless steel bioreactors can be used with a variety of impeller types like Rushton, marine or pitched-blade impellers whereas Xcellerex SUB bags include 3-4 pitched-blade impellers at a 40° pitch. SUB bags are pre-sterilized by gamma irradiation and can be typically used to remove sterile by welding sterile sample containers whereas steam sterilization is required for stainless steel vessels increasing the ease of using SUBs.

Scalability of a Single-use Bioreactor Platform for Biopharmaceutical Manufacturing

Increasing adoption of single-use technologies for bioprocessing along with higher titers from cell culture bioreactor processes has allowed clinical and even commercial manufacturing to be successfully performed in 2000 L-scale single-use bioreactors. Several biopharmaceutical manufacturers have successfully adopted single-use bioreactors for production. However, information about process scalability from glass bioreactors to 2000 L single-use bioreactors for different types of CHO cell lines is not widely available. Here we provide an overview of the key differences between single-use and conventional stainless steel bioreactors, and highlight factors that are employed while scaling-up from small-scale glass bioreactors to 2000 L-scale single-use bioreactors. Several case studies focusing on process performance across scales into single-use bioreactors are provided. This analysis confirms that the 2000 L-scale single-use bioreactor system can be robustly employed for biopharmaceutical manufacturing.

Abstract

Niket Bubna, Cameron T. Phillips, Sigma S. Mostafa and Abhinav A. Shukla KBI Biopharma, Durham, NC

Single-use and Stainless Steel Bioreactors

Factors for Scale-up to 2000 L-Scale Bioreactors

References

Case Studies Across Bioreactor Scales

Conclusions

Acknowledgments

Case Study I – CHO-K1 Cell Line

Case Study II – CHO-S Cell Line

Case Study III – CHO-DG44 Cell Lines (Cell Line A and B)

We would like to thank Sigma and Abhinav for their support and guidance; and express our gratitude to Brian Baker, Lynwel Cunanan, Bryan Howarth, Kathryn Olson, Shaunak Uplekar and Jingshu Zhu for their help.

Comparison between two CHO-DG44 cell lines is shown above (Cell Line A and B from the same vendor). This comparison demonstrates a case where variable cell culture performance was observed in Cell Line A during the first attempt to scale-up in a 200 L-scale SUB bag (red line), but not in Cell Line B. Four process modifications (no change to raw materials or consumables) were implemented to the Cell Line A bioreactor process, resulting in improved cell growth and productivity. The same changed were applied to Cell Line B ensuring seamless scale-up from 3 L to 200 L-scale.

An early-stage cell culture process was transferred from a client. This process first tested at 15 L-scale showed comparable cell culture performance, but required a modification to the day of harvest to achieve consistent product quality results. A strategy to initiate harvest depending on the % acidic species was successfully implemented at 50 L and 2000 L-scale.

This case study illustrates an expedited model of process development, where gene synthesis to release of the first batch of bulk drug substance purified from a 2000 L-scale cGMP bioreactor run was achieved in less than 14 months. Platform cell culture and purification processes were successfully applied to optimize the final process using a stable pool of cells. Process performance was confirmed in 3 L-scale glass bioreactor using Top 3 Clones before scaling up in 200 L and 2000 L-scale SUBs using the Top Candidate Clone.

Parameter XDR-50 SUB XDR-200 SUB XDR-2000 SUB

Impeller Diameter 0.2159 m 0.2159 m 0.4191 m

Impeller Power Number 1.50 1.15 0.72

Pitched-blade Impeller 3 blades at 40° 3 blades at 40° 4 blades at 40°

Turn-down Ratio 2.2:1 5:1 5:1

Aspect Ratio 1.5:1 1.5:1 1.5:1

Parameter Xcellerex Single-Use Bioreactor Stainless Steel Bioreactor

Sterilization Pre-sterilized Bags (γ irradiated) CIP and SIP

Culture Vessel Single-use LDPE Bag Stainless Steel Vessel

Agitator Bottom-mounted pitched-blade Top/Bottom mounted impellers (with or without baffles)

Gas Supply Sparge discs (with or without open-pipe)

Ring sparger, Microsparger, etc.

Sample Port Welding sterile bags SIP

Rushton Impeller Marine Impeller Pitched-blade Impellers (including sparge discs)

Stainless Steel Bioreactors Xcellerex Single-Use Bioreactors

Cell culture processes developed in small-scale glass bioreactors were successfully scaled to single-use bioreactors ranging from 50 L to 2000 L for several CHO cell lines. Scale-up to larger bioreactor volumes was implemented by maintaining power input per volume and VVM across different scales. Results in four CHO cell lines variants show that cell growth, metabolite levels and productivity can be reproduced in large-scale SUBs. We have also developed process steps to overcome suboptimal cell growth in single-use bioreactors as observed in Cell Line A (Case Study IV). Based on the scalability of this single-use bioreactor platform, it will be used for commercial manufacturing in the near future.

Chinese Hamster Ovary (CHO) cell lines are widely used in biopharmaceutical processing to produce monoclonal antibodies and therapeutic proteins. Although clinical or commercial production occurs in large-scale bioreactors, process development and optimization is performed at much smaller scales, in bioreactors ranging from milliliters (microbioreactors) to a few liters (bench-scale bioreactors). Several factors, including bioreactor geometry, agitation rate and gassing strategy must be considered when increasing scale of operation. Agitation Rate • Homogenous mixing is important to provide uniform nutrient, oxygen concentration, pH and

temperature around the cell – improper mixing will develop gradients and result in suboptimal performance

• Limit to maximum mixing in mammalian cells due to cell physiology – lack of cell wall and cell diameter – leads to increased sensitivity to shear forces

• Power input per volume is commonly used to normalize the agitation speed based on the power provided to the impeller. This method is used for scale-up of most mammalian cell culture processes at KBI Biopharma.

Gassing Strategy • Important variables include sparge type, sparge hole size and gas flow rates • A ratio of volumetric gas flow to liquid volume, vessel volumes per minute (VVM), can also be used to scale up gas flow rates. This method is arguably better since it depends on the actual liquid volume in the bioreactor instead of an area which will not change. This allows one to operate at multiple volumes in one vessel while changing flow rates based off the volume.

Several different strategies can be used for scale-up • Maintain bioreactor geometry, P/V, and VVM across scales – Used at KBI Biopharma to develop

scalable processes. • Maintaining bioreactor geometry, kLa, and superficial gas flow – This method prioritizes oxygen

transfer over mixing, using constant kLa to determine the agitation speed. A similar method replaces superficial gas velocity with constant tip speed or shear rate. This ensures the cells are subjected to the same shear stress at all scales.

𝑃𝑃𝑉𝑉

=𝑁𝑁𝑝𝑝𝑁𝑁3𝐷𝐷𝑖𝑖5𝜌𝜌

𝑉𝑉 𝑉𝑉𝑉𝑉𝑉𝑉 =

𝑄𝑄𝐺𝐺𝑉𝑉

where, Di = Impeller diameter [m] N = Agitation speed [s-1] Np = Impeller power number [-] P = Power [W] S = Cross-sectional area [m2] V = Volume [L]

where, QG = gas flow rate [L/min] V = Volume [L] VVM = Vessel volumes per minute [min-1]

Vial Inoculum

Expansion in Shake Flasks

Inoculum Expansion in Wave Cellbag

Inoculum Expansion in XDR-200 SUB

Production Run in XDR-2000 SUB

Clarification using POD

Depth Filters

Typical Cell Culture Process Flow for 2000 L-Scale cGMP Manufacturing at KBI Biopharma

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15 L-Scale 3 L-Scale 3 L-Scale

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Lact

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Lact

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Days2000 L-Scale 200 L-Scale 50 L-Scale3 L-Scale 3 L-Scale 3 L-Scale

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Days100 L-Scale 100 L-Scale 100 L-Scale3 L-Scale 3 L-Scale 3 L-Scale

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Days100 L-Scale 100 L-Scale 100 L-Scale

3 L-Scale 3 L-Scale 3 L-Scale

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Cell Line A Cell Line B

1. Shukla, A. and Gottschalk U. (2013) Single-use disposable technologies for biopharmaceutical manufacturing. Trends Biotechnol. 31, 147-154

2. Eibl, D. et al. (2011) Single-Use Bioreactors – An Overview. In Single-Use Technology in Biopharmaceutical Manufacture (Eibl, D. and Eibl, R., ed.), pp. 33-48, John Wiley & Sons

3. Yang, J. et al. (2007) Fed‐batch bioreactor process scale‐up from 3‐L to 2,500‐L scale for monoclonal antibody production from cell culture. Biotechnol. Bioeng. 98, 141-154

4. Dreher, T. et al. (2014) Design space definition for a stirred single‐use bioreactor family from 50 to 2000 L scale. Eng. Life Sci.14, 304-310

Case Study IV – CHO/dhFr- (Client’s Proprietary Cell Line) Rapid process development (4 months) using the Top Clone resulted in a robust process with 2.5-fold increase in bioreactor titer. Top Clone was utilized for a feed screening experiment and optimization of pH control range. Final process from 3 L-glass bioreactor was scaled to 200 L single-use bioreactor prior to process transfer for clinical manufacturing at 2000 L-scale. Cell growth, productivity and metabolite levels were similar in all three scales.

Viab

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2000 L-Scale 2000 L-Scale 2000 L-Scale2000 L-Scale 2000 L-Scale 200 L-Scale3 L-Scale 3 L-Scale 3 L-Scale3 L-Scale 3 L-Scale

Gluc

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Days2000 L-Scale 2000 L-Scale 2000 L-Scale2000 L-Scale 2000 L-Scale 200 L-Scale3 L-Scale 3 L-Scale 3 L-Scale3 L-Scale 3 L-Scale

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Days2000 L-Scale 2000 L-Scale 2000 L-Scale2000 L-Scale 2000 L-Scale 200 L-Scale3 L-Scale 3 L-Scale 3 L-Scale3 L-Scale 3 L-Scale

Prod

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Days2000 L-Scale 2000 L-Scale 2000 L-Scale2000 L-Scale 2000 L-Scale 200 L-Scale3 L-Scale 3 L-Scale 3 L-Scale3 L-Scale 3 L-Scale

Lact

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Days2000 L-Scale 2000 L-Scale 2000 L-Scale2000 L-Scale 2000 L-Scale 200 L-Scale3 L-Scale 3 L-Scale 3 L-Scale3 L-Scale 3 L-Scale

Amm

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Days2000 L-Scale 2000 L-Scale 2000 L-Scale2000 L-Scale 2000 L-Scale 200 L-Scale3 L-Scale 3 L-Scale 3 L-Scale3 L-Scale 3 L-Scale