High throughput bioreactor mimetic in l d l t t d l tearly and late stage process development

American Chemical Society – Biochemical Technology (BIOT) Division245th – ACS National Meeting, New Orleans, LA

Shahid Rameez, Ph.D.Shahid Rameez, Ph.D.Scientist I, Process Development

KBI Biopharma Inc, Durham, NCp

Overview

Ambr SystemBioreactor Mimetic

In process

Mimetic

KBI Evaluation

pActivities

&Design Space

Evaluation

Experimental Design in Early and Late C t l St t iy

Stage Process Development

Control Strategies

A key bottleneck in biopharmaceutical development has been they p prapid development of robust and scalable manufacturing processes thatcan permit accelerated progress of products into clinical trials.

Innovations in this area can have a very significant impact on theoverall economics of biopharmaceutical drug development byd i h i i k h h li idecreasing the time it takes to reach the clinic.

Mammalian cell culture processes typically have the longestp yp y gexperimental duration with 2-3 weeks being a typical duration for theproduction bioreactor step with additional time spent on the seedculturescultures.

Shake flasks provide the capability to perform high throughputexperiments, but with an inability to control process parameters likeexperiments, but with an inability to control process parameters likeagitation rate, dissolved oxygen (DO) and pH.

Th t l h l h d l i b t ffi i t These parameters play a huge role when developing a robust, efficientcell culture process which dictates the product quality and yield.

Moreover, reproducibility and scalability of process and cultureperformance is a pre-requisite for successful and efficient processdevelopment at a scale down level.development at a scale down level.

At KBI we did case studies evaluating the ambr™ system (ana tomated micro scale bioreactor s stem) to establish the roleautomated micro-scale bioreactor system) to establish the rolethis system could play in accelerating biotech drug development.

Automated Miniaturized Bioreactors

1mL tips

ambrTM Technology

1mL or

LiquidHandler

4mL tipsHandler

Used Tips Discard

Culture stations; each holding 12 bioreactors

ambrTM Deck Layout

Culture stations

1ml tips4ml tips4ml tips

24 deep-well plate Tip box lid

Plate lid

The Vessel

Vessel Cap:

In-line filter on gas supply

DO sensor

on gas supply

ImpellerpH sensorp

Culture Station Nomenclature

Culture station 1 (CS1) Culture station 2 (CS2)

CS1

-1

CS1

-2

CS1

-3

CS1

-4

CS1

-5

CS1

-6

Culture station 1 (CS1)

CS2

-1

CS2

-2

CS2

-3

CS2

-4

CS2

-5

CS2

-6

Culture station 2 (CS2)

7 8 9 10 11 12 7 8 9 10 11 12

CS1

-7

CS1

-8

CS1

-9

CS1

-1

CS1

-1

CS1

-1

CS2

-7

CS2

-8

CS2

-9

CS2

-1

CS2

-1

CS2

-1

Id l ki l i 13mL W ki l i 11 15mLIdeal working volume is 13mL; Working volume range is 11 – 15mL

Things to consider:

– Feed strategy and Sampling strategyFeed strategy and Sampling strategy

– Low volumes – gas entrainment / vortexing

– High volumes – Limits kLa.

PART 1: Reproducibility for Results and Key Observations during Cell Culture Process Development

The processes evaluated in ambrTM were previously developed from a rigorouscell culture process development performed in classical bioreactors of variousscales. The process were successfully carried in bioreactors across various scales: 2L,10L d 200L10L and 200L.

This study aimed at studying the reproducibility of the key observations of theprocesses in ambrTM. Thus a reverse engineering approach was adopted, where wewere cognizant about the outcomes from most of the experiments as far asinducing process variations was concerned.

The reproducibility of key historical results in ambrTM would corroborate towardsits capability as a high throughput bioreactor mimetic in cell culture processdevelopment.

K Ob i f Hi i l D

Case Study 1: Reproducibility evaluation for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary (CHO) cell line.

Key Observations from Historical Data:• Temperature Shift during the cell culture process was found to be the most important

process factor to regulate the productivity of the antibody titer.

• The CHO cell line performed better at lower pH set point of 6.85 as compared to pHset 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 for feed.

• A highly basic and a critical feed, referred here as FDX, had to be added without pre-neutralization with acids to avoid osmolality increase in cultures. Thus, a better controlhad to be established in the bioreactors to control the pH drift with addition of FDX.

h h d b b h d hThis was achieved in classical bioreactors by tuning the PID controllers and withregulation in cascade feedback gassing of CO2 and Air.

Results: Both lower pH set-points and Temperature Shift showed higher cell growth, bettercell viabilities. DO as suspected at negligible effect on cell growth and viability.

Time courses for viable cell growth and viability for recombinant CHO cell line with changing(A) Process pH (B) Temperature (C) Dissolved Oxygen (DO) levels and (D) Feeding Strategies. Theexperimental data shows an average of 2-3 vessels in the ambr 24. The error bars show the standarddeviationdeviation.

Results: Both lower pH set-points and Temperature Shift showed higher cell titers. Asobserved historically for this process, Temperature shift was found to be the most importantprocess factor to regulate the productivity of the antibody titer.

The ambrTM system can be used as a high-throughput platform to make keyprocess decisions during the early process development phase ofbi h i l d lbiopharmaceutical development.

PART B: Scalability Assessment in Cell Culture Process Development

Case Study 2: Comparison across scales for the production of a monoclonal antibody in a recombinant CHO cell line.

Ambr (n = 3). 2L (n = 1).

10 ( 4)10L (n = 4). 200L (n = 1).

• Harvest Titers within 1 5 1 7 g/L• Harvest Titers within 1.5 -1.7 g/L across all scales.

Comparison of time courses for viable cell growth and viability for recombinant CHO cell line in ambrTM and otherscales bioreactors: 2, 10L Glass bioreactors and 200L disposable bioreactor.p

Case Study 3: Comparison across scales for the production of a protein molecule in arecombinant Chinese Hamster Ovary (CHO) cell line.

Results: The cell growth, cell viability and cellviabilities were comparable between ambrTM,10 and200L Bioreactors200L Bioreactors.

Case Study 3: This protein molecule had two Isodimers (A and B). The levels of Isodimers A and B were a product quality attribute.

Results: Ratio of Isodimers Aand B were similar (± 5% ofmean values) across ambrTM , 10and 200L Bioreactors.

The process decisions and results from ambrTM were reproducible to theresults in other scales bioreactors.

Both the case studies (with antibody and a non antibody) demonstrate theutility of the ambr™ system as a high throughput system for cell cultureprocess development.p p

PART C: Control for process pH and DO during Cell Culture Process Development

pH control in ambrTM is established using theautomated liquid handler based base additionswhen pH drops below the pH set pointwhen pH drops below the pH set point.

When the pH exceeds the pH set point, theCO2 flow rate increases to establish control onthe pH drift.

CO2: 0 - 1.24 mL/min. Delivered on demand to control pH.O2 : 0 - 1.24 mL/min. Delivered on demand to control dissolved oxygen.N : 0 1 24 mL/min Flow rate is constantN2 : 0 - 1.24 mL/min. Flow rate is constant.

Online profiles for process pH (top Online profiles for process pH (topfigure) and DO (bottom figure) levelsduring the culture duration for CHO cellline expressing a recombinant antibodyin ambrTM.

The spikes in the DO profilescorresponded to bioreactor samplingcorresponded to bioreactor sampling,Liquid additions and Sampling.

All these disturb the headspace andl h ki l Th i falter the working volume. The time for

the DO traces to equilibrate to setpointafter such manipulations would dependon the controller setup.p

Case Study 4: Artificial perturbations in pH and DO (by adding a basic feed and changing DOset points respectively) during production of an antibody molecule in a recombinant CHO cellprocess. Through adjustments to the PID control loop and gas flow rates the capability of

b ™ t l t dambr™ system was evaluated.

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 > 2folds asopposed to default manufacturer values , the pH drifts were reduced by 23 and 47 % of initialvalue, respectively.value, respectively.

Results: The DO set points were changed to 80% from 20 and 40%, respectively andchanged back to original values The level for DO was maintained at 80% for duration of 6changed back to original values. The level for DO was maintained at 80% for duration of 6hours and returned to original set points 20 and 40% in ≈ 90 and 120 mins, respectively.

Th p bilit f ind in d i ti n n h lp in d i nin r t The capability of inducing deviations can help in designing worst-caseexperiments. It enables to test operating limits with respect to particularkey operational parameters (DO, pH) in a process.

The combination of pH and DO control and an automated liquid handling system inambrTM system overcomes major limitations of conventional small-scale cultures vessels

Conclusions

ambr system overcomes major limitations of conventional small scale cultures vesselsespecially shake flasks.

The single-use, pre-calibrated, and instrumented vessels used in ambrTM system providesa platform for high-throughput in cell culture process development while mimicking astirred-tank bioreactor environment.

The reproducibility of key observations observed in historical process developmentT e ep od c b ty o ey obse vat o s obse ved sto ca p ocess deve op e tdemonstrated that ambr™ is capable of providing predictive results under bioreactorrelevant process conditions.

R d ibili l bili d h bili f h d b i h Reproducibility, scalability and the ability of the system to respond to perturbations showambrTM to be adequate to consider this system for early and late stages of cell cultureprocess development.

The studies at KBI aimed to demonstrate the utility of the ambr™ system as ahigh- throughput bioreactors that can offer the realistic possibility of decreasing theprocess development time for investigational biopharmaceuticals to reach the clinic.

Process CharacterizationCommercial Process Process Cell Line Discovery Manufacturing

Biopharmaceutical Development Process.

Characterizationand ValidationDevelopmentDevelopmentDevelopment

yStage

RAPID PRODUCT DEVELOPMENT AT KBI

Manufacturing

ambrTM

Process Development(Design Space & Optimization)Cell Line DevelopmentDiscovery Stage Manufacturing

• Platform Downstream Processes• High-throughput Resin Screening

• Single - Use TechnologyTM The combination of methodologies such as ambrTM, Platform Downstream

Processes, High-throughput Resin Screening and use of Single-use technologycan significantly shorten the window for process development and

f t imanufacturing.

Acknowledgements

• Joe McMahon President and CEO • Abhinav Shukla, Ph.D. VP, Process Development and Manufacturing• Sigma Mostafa Ph D Director Process DevelopmentSigma Mostafa, Ph.D. Director, Process Development• Haiou Yang, Ph.D. Scientist II, Process Development• Christopher Miller Scientist II, Process Development• Anushya Mani Scientist I Process Development• Anushya Mani Scientist I, Process Development• Joe Jirka Product Specialist, TAP Biosystems

Process Development Team at KBI Process Development Team at KBI

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