implementing true pat in bioreactor operations · bioreactor models, have also been developed to...

Bend Research Inc. 64550 Research Road, Bend, OR 97701 USA

Phone: 1-800-706-8655 © Bend Research Inc. 2014

Implementing True PAT in Bioreactor Operations: Gaining Optimized Cell-Level “Observability” and Data-Driven Process “Guidance” by Leveraging On-Line technologies such as Dielectric Spectroscopy and

At-Line Tools such as the Modular Automated Sampling Technology (MASTTM) Platform

Abstract

Conclusions

Authors: C. Pepper,a L. Graham,a B. Downey,a D. Newbold,a J. Germon,b and D. Sullivanb (a Bend Research Inc., b Pfizer Inc.)

Acknowledgments: The authors thank J. Bouressa, N. Jenkins, E. Yu, S. Casnocha, J. Weber, T. Barreira, P. Jeffers, and C. Crowley of Pfizer, and T. Wigle, D. Millard, A. Carroll, N. Glutting, P. Glover, B. Russell, L. Davis, and R. Ray of Bend Research for their contributions to this work.

In implementing process analytical technology (PAT), biopharmaceutical companies are continually striving to gain a fundamental understanding of what is happening to the cells within their bioreactors and the impact that the process has on the cell’s ability to deliver the target product quality. While implementation of on-line tools like dielectric spectroscopy and Raman are helping to provide insight, there is still a gap integrating the data produced by these techniques with off-line measurements such as cell density, viability, metabolite levels, and titer and presenting that data to the end user that gives them real insight into their process. Bend Research Inc., in collaboration with Pfizer Inc. and other major biopharmaceutical companies, is working to advance the Modular Automated Sampling Technology (MAST) platform, the goal of which is to provide a complete data management solution to the end user. The MAST system prototype covers all aspects of data management from providing aseptically collected bioreactor samples to analytical devices, maintaining the raw analytical data in a database and then providing the user with a Graphical User Interface (GUI) to rapidly modulate the data, providing instantaneous guidance. Early studies with this modular sampling platform have demonstrated that automated at-line measurements are representative of parallel manual samples. During these studies, maintenance of sterility has not been an issue. This poster illustrates how the MAST platform is incorporated with on-line analytical technologies to deliver overall cell-level “observability.” For example, as a result of this integration, dielectric spectroscopy measurements can be corrected to estimate total and viable cell density more accurately. From this new level of insight into the operation of the system, scientists can obtain enhanced data-driven “guidance” for key activities like cell-line selection and optimized process operation. With this new technology, the bioprocess industry can make major advances toward advanced real-time testing, predictive control, and overall enhanced bioprocess design and operation.

Recent innovations in on-line measurement (dielectric spectroscopy and Raman) and modular, automated aseptic sampling (MAST) have created the conditions needed for true bioreactor PAT implementation. The MAST system is ideal for use in bioreactors. It demonstrates reliable contamination-free sampling with greater sample consistency and reproducibility when compared to manual samples. This scale-independent, low-cost sampling system is capable of frequent sampling to enable more intensive process-control schemes. Savings in labor and process optimization/efficiency can be expected. The highly efficient Sample Pilot modules also have potential use in disposable systems and downstream applications. The combination of advanced on-line measurement with at-line analysis (e.g., dielectric spectroscopy and Raman) creates a synergistic environment for accelerated process understanding. Additional PAT, coupled with cell-based bioreactor models, have also been developed to enhance guidance. This emerging process-development methodology holds promise to shorten development timelines and deliver a higher-quality process that significantly reduces the cost of goods.

Delivering Biotherapeutics: Our Mission Deliver Innovative and Transformative

Technologies & Services … To Achieve Target Quality Proteins … through Science & Engineering

Sample Pilot SP100

Fixed Reactor Installation - Steam or Liquid Sanitization

Sample Pilot SP200

Bench-Scale and SUB Reactors - Liquid Sanitization

Time-series plots of Raman models and NovaFlex data collected through the MAST Sample Pilot. By enabling samples to be collected at frequent, regular intervals without an operator present, we were able to increase our off-line data without any additional labor. Those data were used to build robust process monitoring models and develop the Raman system for laboratory use. The Sample Pilot is an enabling existing technology for implementation of PAT applications.

Raman Data Correlated to Nova Results Collected At-Line Using the MAST Sample Pilot (SP100)

Dielectric Spectroscopy (DS) Data Correlated to Off-Line Caspase Activation

Measurements Enable On-Line Monitoring of Cell Population State

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As cell viability drops in response to staurosporine addition, transformations in the cell lead to variations in capacitance. By scanning over a frequency range, it was discovered that distinct cell populations could be distinguished during apoptosis. These observations in the capacitance spectra were related to off-line measurements of Caspase 3 activation to provide an on-line measure of cell health in the bioreactor.

“Cell-Level”

Example schematic showing potential for sampling from multiple bioreactors to enable “at-line” analyses (in conjunction with “in-line” technologies (e.g., Dielectric Spectroscopy and Raman); Potential application: Process Control

Good comparison shown between manual and MAST samples taken from the 500-L reg tox pilot-scale runs

MAST Sample Pilot (SP100) on a 30-L Bioreactor

Pilot bioreactor with two Raman probes installed

“Process-Level”

“On-Line” Technologies “At-Line” Technologies

Enabling Cell & Protein Level “Observability” “Guidance”

Cell Separation and / or

In-house testing shows excellent consistency between manual and MAST samples taken from bench- scale cell culture runs

Location

Scale /

Sample Pilot

Model

Situation Number

of Runs

Total

Accumulated

Duration (days)

Total

Samples

Taken

Outcome

Bend

Research

Laboratory

SP200 Cell Culture 2 30 69

No contamination. Results

consistent with manual

sampling

Bend

Research

Laboratory

SP200

Media

Challenge 3 98 >1141 No contamination

Pfizer Pilot

Plant

30-L Reactor




sampling

Pfizer Pilot

Plant

130-L Reactor




sampling

Pfizer Pilot

Plant

500-L Reactor




sampling

Company

A

Pilot-Scale




sampling

Company

B

Pilot-Scale

SP200

High Cell

Density

Challenge

6 6 244

Overnight and repetitive

sampling with no clogging or

fouling

Company

B

Pilot-Scale




sampling

Company

C

Pilot-Scale

SP100 Microbial 2 10 10



sampling

Company

D

Laboratory




sampling

Company

E

Laboratory

SP200 Cell Culture 1 7 4 No contamination

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