Scalability of a Single-Use Bioreactor Platform for Biopharmaceutical Manufacturing

Niket BubnaPrincipal Scientist, Process Development, KBI Biopharma

Durham, NC

Presented at PepTalk 2017: San Diego, CASingle-use Technologies And Continuous Processing (Advancing Bioprocessing Through Technological Innovation)Risk Mitigation Strategies For Single-use Technologies

Jan 2017PepTalk 2017

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2013-14 2015 2016

New Products Mfg Batches

Cell Culture Manufacturing in KBI Biopharma (Durham, NC)

Reference: Medicines in Development (Biologics). 2013 PhRMA Report

• New biopharmaceuticals being developed at a record rate• Rapid development and manufacturing for FIH studies is

needed to support the rate of discovery• Tox and FIH studies require smaller quantity of product

• Commercial needs can be met with 2000 L-scale bioreactors leveraging high titer cell culture processes

• Need for a readily available plan and plant• Plan includes process, equipment and supply chain • Equipment and supply chain are available• Scalable SUB platform needed


Reference: Shukla, A., Mostafa, S., Wilson, M., Lange, D. Vertical Integration of Disposables in Biopharmaceutical Drug Substance Manufacturing, Bioprocess International, 10(6), 34-47, 2012.


Typical Cell Culture Manufacturing Unit Operations at KBI (Durham, NC)

• Single-use bioreactors are being used for clinical manufacturing and even commercial manufacturing

• Clinical manufacturing» Wave for ~2 decades» Stirred tank 2000 L-scale since 2009

• Commercial manufacturing» Amgen, Singapore

• Multiple SUB platforms are available• HyClone HyPerforma• Sartorius BIOSTAT STR• Millipore Mobius


• Minimal cleaning requirements• Quick turnaround leading to higher plant capacity• Flexibility of manufacturing multiple products• Fewer requirements for changeover• Reduced risk of cross-contamination



• Agitation Rate• Homogenous mixing is important to provide uniform environment

around the cell• Power input per volume is commonly used to normalize agitation

rate based on the power input to the impeller

• Gassing Strategy• Variables: Sparge type, sparge hole size and gas flow rate• Use of VVM (vessel volumes per minute) or the ratio of volumetric

gas flow to the liquid volume is used; it depends on the actual culture volume, allowing operations at multiple volumes

• Combination of three key factors:• Power input per volume• Volumetric gas flow rate• Bioreactor geometry


𝑃𝑃𝑉𝑉

=𝑁𝑁𝑝𝑝𝑁𝑁3𝐷𝐷𝑖𝑖5𝜌𝜌

𝑉𝑉

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

𝑉𝑉𝑉𝑉𝑉𝑉 =𝑄𝑄𝐺𝐺𝑉𝑉

where,QG = gas flow rate [m3/s]V = Volume [L]VVM = Vessel volumes per minute [min-1]

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



Project Cell Line Type Cell Line Vendor Basal Medium Doubling Time (hours)

Forward Processing VCC (10^6 cells/mL)

Passage Duration

Can Platform Approach Be Used?

A CHO-DG44 A & KBI OptiCHO / Dynamis 25-35 2-3 3-4 days No

B CHO-K1 B & KBI ProCHO 5 21-24 1.7-2.5 3 days No

C-F CHO-S A & KBI Dynamis 14-20 3-7.5 3-4 days Yes

G Proprietary C BalanCD Growth A 18-21 3.5 3 days Yes

H CHO-DG44 D BalanCD Growth A 16-17 4 3 days Yes

I CHO-GS E FortiCHO 25-40 None 2-4 days No

J CHO-GS F CD CHO 20-24 3 3 days No

K Proprietary C BalanCD Growth A 15-20 2-6 3 days Yes

L CHO-GS G Client Medium 37-42 3 3-4 days No

M CHO-GS F OptiCHO / FortiCHO 22-23 3 3 days Yes

N Proprietary(CHO-K1) H OptiCHO /

PowerCHO-GS 20-30 3.5 4 days No

O Proprietary C BalanCD Growth A 17-21 3.5 3 days Yes

P CHO-DG44 E ExCell CHO 30-40 1.5 2-3 days No

Q-S CHO-DG44 A & KBI OptiCHO 20-30 2.5-4.5 3-4 days Yes

T CHO-DG44 D ExCell ACF CHO 21-24 4 3-4 days No

U Proprietary I OptiCHO 25-35 1.5-5 3-4 days Yes




Rapid process development to support IND filing & FIH Studies• Top Candidate Clone to final cell culture process in 3.5

months• Applied platform process (medium-feed and process parameters)• Required feed media change to support increase in titer

• Process scale-up and cGMP manufacturing completed in 5 months

• Right First Time without a non-GMP Engineering Run• 5 cGMP runs at 2000 L-scale carried out successfully

Proprietary CHO Cell Line


Viab

le C

ell C

ount

(10^

6 ce

lls/m

L)

Days

3 L-Scale (n=5)

200 L-Scale (n=1)

2000 L-Scale (n=5)

mAb

Tite

r (g/

L)

Days

3 L-Scale (n=5)

200 L-Scale (n=1)

2000 L-Scale (n=5)

Proprietary CHO Cell Line



Process Transfer from a Client for a mAb• Final cell culture process transferred from client CMO

• Gaps were identified in transferred process• Development runs were necessary to establish process conditions• Production bioreactor harvest day chosen based on product quality

• Re-optimized process was scaled-up to 2000 L-scale• Successfully implemented strategy of harvesting bioreactor based

on product quality target

CHO-K1 Cell Line


CHO-K1 Cell LineVi

able

Cel

l Cou

nt

Days2000 L-Scale 2000 L-Scale 50 L-Scale

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

Gluc

ose

Days



Amm

onia

Days



Prod

uct C

once

ntra

tion



% A

cidi

c Spe

cies

Days


Lact

ate

Days



Prod

uct Q

ualit

yAt

tribu

te



Application of Platform Cell Culture Process for 4 mAbs• Expedited process development using an un-optimized

platform process• Pre-determined medium-feed combination and bioreactor

process parameters• Key objective was speed and consistency rather than quantity of

product

• Process transferred to manufacturing scale without non-GMP Engineering Run for all 4 mAbs

• Gene to IND filing in less than 14 months

CHO-S Cell Line

• HCCF supply runs for purification and analytical method development are typically carried out in 50 L SUB using a stable pool of cells and a platform cell culture process

• This approach ensures that compatibility of cell line and process with SUB bags is tested early in development

• Wave Cellbags for inoculum expansion• Xcellerex SUB bags for production-stage


CHO-S Cell Line

Stable Pool

HCCF Supply Run (50 L SUB)

Clone SelectionProcess Optimization

Platform Process Assessment

Process Confirmation (50 L SUB)

Scale-up (200 L SUB)

Expedited Cell Culture Development

Top 3 Clones

Top Clone

SUB performance evaluation

Wave or Seed bioreactor performance evaluation


Viab

le C

ell C

ount

Days

2000 L-Scale 200 L-Scale 50 L-Scale3 L-Scale 3 L-Scale 3 L-Scale

Prod

uct C

once

ntra

tion

Days


Gluc

ose

Days


Lact

ate

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

CHO-S Cell Line


Development and Application of Platform Cell Culture Process for Recombinant Vaccine Proteins

• Challenges during process development:• Low productivity & stable-clone selection• Cell viability drop during production-stage• Product cleavage

• Challenges during initial scale-up attempts (Cell Line A):• Lack of scalability from glass bioreactor to SUBs

» Cell growth and cell viability were significantly divergent• Challenges were overcome; increasing robustness and reproducibility of the

platform process (shown by Cell Line B)


CHO-DG44 Cell Line


Viab

le C

ell C

ount

Days

200 L-Scale 200 L-Scale 200 L-Scale200 L-Scale 15 L-Scale 3 L-Scale3 L-Scale

Gluc

ose

Days200 L-Scale 200 L-Scale 200 L-Scale200 L-Scale 15 L-Scale 3 L-Scale3 L-Scale

Cell V

iabi

lity

Days

200 L-Scale 200 L-Scale 200 L-Scale200 L-Scale 15 L-Scale 3 L-Scale3 L-Scale

Lact

ate

Days200 L-Scale 200 L-Scale 200 L-Scale200 L-Scale 15 L-Scale 3 L-Scale3 L-Scale

Cell Line A CHO-DG44 Cell Line

• Observed poor cell growth and drop in cell viability upon scale-up in single-use bioreactor bag

• Two key hypotheses• Leachable/extractables• Adsorption of nutrients from basal medium

• Steps taken to mitigate both potential risks• Pre-treatment of bioreactor bag (wash) prior to medium addition• Addition of feed media and supplements to overcome depletion

of nutrients


CHO-DG44 Cell Line


Viab

le C

ell C

ount


Prod

uct C

once

ntra

tion



Gluc

ose

Days


Lact

ate


Cell Line B CHO-DG44 Cell Line

• Single-use bioreactor scale-up platform has been successfully developed and implemented for a variety of cell lines, cell culture processes and biopharmaceutical products

• This single-use bioreactor platform ensures that process scale-up remains smooth and seamless; and cell culture process development can focus on key product quality or titer requirements

• KBI Biopharma is uniquely placed with the right knowledge base and experience to serve needs of the biopharmaceutical industry


• Abhinav Shukla• Sigma Mostafa• Cell Culture Process Development

• Brian Baker• Lynwel Cunanan• Bryan Howarth• Kathryn Olson• Shaunak Uplekar

• Analytical Development & Testing• Jimmy Smedley• Amanda Hoertz’s Group• Nate Oien’s Group


• Manufacturing & Manufacturing Sciences

• Carnley Norman• Aaron Anders• Ian Bales• Seth Moye• Ronnie Nichols

Health & Medicine

Scalability of a Single-Use Bioreactor Platform for Biopharmaceutical Manufacturing