INTRODUCTION

Decreasing the timeframe for cell culture process development has been a key goal towards accelerating biopharmaceutical development.

Automated Micro-scale Bioreactors (ambr™) is an advanced micro bioreactor system with miniature single-use bioreactors with a 9-15mL

working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its

performance for the production of a monoclonal antibody and a non-antibody molecule in recombinant Chinese Hamster Ovary (CHO) cell

lines.

The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3L, 15L and 200L stirred tank

bioreactors. Moreover, changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which

were in good agreement to historical data from the larger scales. ambr™ was found to successfully reproduce variations in temperature, dissolved

oxygen and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to

perturbations in pH and dissolved oxygen through adjustments to the PID control loop. Overall, the studies demonstrate the utility of the

ambr™ system as a high throughput system for cell culture process development.

High-throughput Miniaturized Bioreactors for Cell Culture

Process Development: Reproducibility, Scalability and Control. Shahid Rameez, Sigma S. Mostafa, Anushya Mani, Haiou Yang, Christopher Miller and Abhinav A. Shukla

KBI Biopharma, 1101 Hamlin Road, Durham, NC 27704

ambrTM SYSTEM

ABILITY TO MAKE KEY PROCESS DECISIONS FASTER DURING CELL CULTURE PROCESS DEVELOPMENT

CASE STUDY I: Reproducibility of key historical results in ambrTM for the production of a monoclonal antibody in a CHO cell line.

Combination of pH and DO control and an automated liquid handling system in ambrTM system overcomes major

limitations of conventional small-scale cultures vessels especially shake flasks.

The single-use, pre-calibrated, and instrumented vessels used in ambrTM provides a platform for high-throughput in cell

culture process development while mimicking a stirred-tank bioreactor environment.

Results: Both lower pH set-points and Temperature Shift showed higher cell growth, better cell viabilities. DO as suspected had minimal effect on cell

growth and viability.

Processes at lower pH set-points and with Temperature Shift showed higher cell titers. As observed historically for this process, Temperature

shift was found to be the most important process factor to regulate the productivity of the antibody titer.

The ambrTM system can be used as a high-throughput platform to make key process decisions during the early process

development phase of biopharmaceutical development.

Time courses for viable cell growth, viability and antibody titer for recombinant CHO cell line with changing (A) Process pH (B) Temperature (C) Dissolved Oxygen (DO) levels and (D) Feeding Strategies. The experimental data shows an average of 2-3 vessels in the ambrTM system. The error bars show the standard deviation.

The scope of the study was to evaluate if key observations from historical data in other scales bioreactors could be reproduced in ambrTM

system. Some Key Observations from Historical Data were:

Temperature Shift during the cell culture process was found to be the most important process factor to regulate the productivity of the

antibody.

The CHO cell line performed better at lower pH set point of 6.85 as compared to pH set point of 7.00

Feeding intermittently had shown to regulate growth and productivity in the process. Intermittent feeding had showed better results than just

Day 0 additions of feeds.

Based on process knowledge, DO levels (20-50%) were expected to have minimal effect on cell growth and viability.

Both the case studies (with antibody and a non antibody) demonstrate the utility of the ambr™ system as

a high throughput system for cell culture process development.

Comparison of time courses for viable cell growth and viability for recombinant CHO cell line in ambrTM and other scales bioreactors: 3, 15L glass bioreactors and 200L disposable bioreactor.

The aim was to compare growth profiles, titer and product quality to other scales 3, 15 and 200L.

Bioreactor System Vendor Maximum Working

Volume (L)

Catalog # / Model

3L Applikon 2.50 Z611000310

15L Applikon 15.00 Z611001510

200L Xcellerex 200.00 888-0151

Table 1: Bioreactors used during the historical cell culture process evaluated in this study.

COMPARISON FOR CELL GROWTH, TITER & PRODUCT QUALITY WITH BIOREACTORS ACROSS VARIOUS SCALES

CASE STUDY II: Production of a monoclonal antibody in a recombinant CHO cell line in ambrTM and other scales bioreactors

CASE STUDY III: Production of a non-antibody in a recombinant CHO cell line in ambrTM and other scales bioreactors

Comparison of time courses for viable cell growth, viability and titers for recombinant CHO cell line in ambrTM and other scales bioreactors: 15L glass bioreactors and 200L disposable bioreactor.

This protein molecule had two Isomers (A and B). The levels of

Isomers A and B were considered as a product quality attribute.

Results:

Ratio of Isomers A and B were similar (± 5% of mean values)

across ambrTM , 15 and 200L Bioreactors.

Results: The cell growth and titers within ± 10% of mean values across all scales.

Bioreactor System Cell Maximum Growth Ratee

(1/d)

Cell-specific Productivity

(pg/cell/d)

ambra 0.159 ± 0.001 16.20 ± 0.02

200Lb 0.150 11.70

15Lc 0.150 10.80± 0.02

3Ld 0.162 10.60

Table 2: Cell culture performance comparison between bioreactor systems (ambrTM, 3 and 15L glass

bioreactors and 200L disposable bioreactor) for Cell-maximum growth rate (1/d) and Cell-specific

productivity (pg/cell/d).

ABILITY TO ACCURATELY AND EFFECTIVELY CONTROL pH AND DO DEVIATIONS

The above figure shows schematic of pH control in ambrTM established using the automated liquid handler based base additions

when pH drops below the pH set point. When the pH exceeds the pH set point, the CO2 flow rate increases to establish control

on the pH deviation.

Online profiles for process pH (top left figure) and DO (bottom left figure)

levels during the culture duration for CHO cell line expressing a

recombinant antibody in ambrTM.

The spikes in the DO profiles corresponded to bioreactor sampling, Liquid

additions and Sampling.

All these operations disturb the headspace and alter the working volume.

The time for the DO traces to equilibrate to setpoint after such

manipulations would depend on the controller setup.

CONCLUSION

The multi-stage nature of process development makes it time and resource intensive. A high throughput scale down

approach to upstream process development is needed to decrease the timeframe from DNA to toxicology studies prior to clinical

entry. The results from the study indicate that ambr™ operated under fed-batch conditions with pH, DO and feed control could

successfully simulate bioreactor culture conditions with highly reproducible results between the replicates. Cell growth, process

capabilities, antibody titer and product quality profiles were assessed and compared to historical classical bioreactor cultivations

and found to be within 5-15% of the historical values.

The reproducibility of key results observed in historical process development demonstrated that ambr™ is capable of

providing predictive results under bioreactor relevant process conditions. The 24 single use vessels provide flexibility to run

larger experimental designs at a time to evaluate feeding regimes, process operating limits and interactions between various

operating parameters. The experiments with pH drift control and excursions in process DO set points demonstrated the process

control capability of ambr™. This system offers the realistic possibility of decreasing the process development time for

investigational biopharmaceuticals to reach the clinic.

CASE STUDY IV: Excursions in process pH and DO to test process robustness and failure limits for process parameters.

Artificial perturbations in pH and DO (by adding a basic feed and changing DO set points respectively) during production of

an antibody molecule in a recombinant CHO cell process. Through adjustments to the PID control loop and gas flow rates

the capability of ambr™ system was evaluated.

Results: The DO set points were changed to 80% from 20 and

40%, respectively and maintained at 80% for duration of 6 hours.

Afterwards, the DO was changed back to original set points 20

and 40%, at the rate of ≈ 0.5% DO/min based on the tuned

controller set up.

Results: Tuning the gas flow limits and proportional gains in

the PID loop of ambr™ system; By changing the

proportional gain by eight folds and CO2 gas limits by 1.25

and > 2 folds as opposed to default manufacturer values, the

pH deviation were reduced by 23 and 47 % of initial value,

respectively.

The capability of inducing deviations can help in designing worst-case experiments for testing process

robustness. It enables to test operating limits with respect to key operational parameters (DO, pH) in a process.

Role of ambr™ in rapid product development throughout the biotech development lifecycle.

a: n = 3, b: n = 1, c: n = 4, d: n = 1.e: Measured from days 0-8.

Tit

er (m

g/m

L)

ambr 15L 200L