BD Resurge™ CD Supplements

BioProcess International
2014 Online Education Series
Send ques)ons to: [email protected] Use and Optimization of
Chemically Defined Media
Supplements in Mammalian Cell
Culture Processes
Elizabeth C. Dodson, Ph.D
R&D Manager
BD Advanced Bioprocessing
BD Biosciences Advanced Bioprocessing
A Global Partner in Bioprocess Development
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Collaborative, trusted partner with global reach
More than 100 years of experience in media and supplements
Proven media and feed design process
Unmatched quality & risk reduction manufacturing
Temse
San Jose
San Diego
Detroit
Baltimore
RTP
Miami
Singapore
Sao Paulo
Today’s Agenda
!  Drivers of successful bioprocess cell culture optimization
!  CHO cell line: History and usage
!  Base media interaction with supplementation
!  Use and Optimization the use of CD supplements
Case Studies will use BD CD Resurge™ as the model CD supplement
!  Case Study 1: Growth and production
!  Case Study 2: Media and supplementation
!  Case Study 3: Feeding strategies
!  Case Study 4: Bioreactor scale-up
Drivers of Successful Bioprocess Optimization
CHO as the preferred host cell line type
!  Which mammalian host cell line?
!  CHO, NS0, PerC6, other (avian, human)
!  Why a CHO (Chinese Hamster ovary) cell line?
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Historical – company platform
Ease of use – suspension, simple to grow
Highly mutagenic – easy to transfect
Resistant to viral infection
Potential protein yield
!  commonly obtain 1-3 g/L
!  often see 3-5 g/L
!  reports of >10 g/L
!  Protein quality – human-like glycosylation patterns
!  Broad availability of choices for media and supplementation designed for CHO
cells
!  Key variants of CHO: CHO-K1, DG44, DXB11
All CHO are not created equal:
Divergence of CHO host cells
CHO-K1: Genomic structure different
than the Chinese hamster originator
Divergence of CHO host cells
Wurm, et al. (2011). First CHO genome. Nature Biotechnology: 29 (8).
What drives biological performance?
Cell Line
Feed
Strategy
Media and
Supplements
Biological
Performance
What drives biological performance?
Cell Line
Feed
Strategy
Media and
Supplements
Biological
Performance
Mammalian Cell Culture Medium
and Supplementation
Vitamins
Amino
Acids
Fatty
Acids
Sugars
Bulk Ions
(Salts)
Trace
Elements
Cell
Culture
Medium
Composition of
Typical Medium and
Supplement
Nucleic
Acids
Basic Components of
Cell Culture Medium
Development Process
BD Resurge™ CD PAK (CD1-CD5)
Innovation
You Can Expand On
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BD Resurge™ Compared to
Other Supplements
Performance of eight commercially available CD supplements were evaluated together
with BD Resurge™ CD1-CD5.
Batch culture studies
All supplements used as directed by manufacturer
3 CHO cell lines in their optimal medium
CHO Line 1 BD Resurge CD1 BD Resurge CD2 BD Resurge CD3 BD Resurge CD4 BD Resurge CD5 Supplement A Supplement B Supplement C Supplement D Supplement E Supplement F Supplement G Supplement H CHO Line 2 CHO Line 3 Highest Titer Signiﬁcant increase Moderate increase No signiﬁcant eﬀect Innovation
You Can Expand On
BD Resurge™
Growth and Protein Yields
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Batch mode, supplements were added on Day 0
Fed-batch mode
•  CD1 and CD2: Day 0, 2 and 5
•  CD3: Day 0, 5 and 7
•  CD4 and CD5: Day 0, 3, 5
Innovation
You Can Expand On
Protein Quality:
N-Glycan Analysis
Type of Base Media/
Supplement CHO 1 ~ Peptone
Type of expression host Cell Culture Process N-‐glycan proﬁle CHO 1 ~ CD1 &CD2
CHO 2 ~ CD3,CD4,&CD5
Case Studies:
How to use and optimize a CD
Supplement
Case Study 1: Growth and production
Case Study 2: Media and supplementation
Case Study 3: Feeding strategies
Case Study 4: Bioreactor scale-up
Case Study 1
CHO Responses to Different CD Supplements
Growth and Production – 2 CHO Lines
Experimental Setup:
Two CHO cell lines expressing mAb
Thirteen (13) CD Supplements
Each cell line is grown in its optimal base medium
Batch culture study
Cell lines supplemented in respective base medium on Day 0
!  Growth and protein production differ for each cell line for each CD supplement.
!  Growth and production levels do not always correlate.
Case Study 1
Summary and Conclusions
!  CHO cell lines do not respond equally to CD
supplementation.
!  A supplement that may work well with one CHO cell
line may not be optimal for another CHO cell line.
!  Growth and production responses don’t necessarily
correlate.
Case Study 2
Media and Supplementation
CD Supplement Performance Dependent on Basal Medium
Experimental setup:
Two (2) CHO cell lines expressing mAb
Three (3) basal media
Five (5) BD Resurge™ CD supplements
1200
600
400
Titer (mg/L)
Titer (mg/L)
1000
200
800
600
400
200
Medium B
Medium C
Medium A
CHO Line 1
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Production levels vary depending on base medium.
CD supplement driven production varies between cell lines.
CD supplement driven production varies across basal media.
Medium B
CHO Line 3
4
2
Control
5
3
1
4
2
Control
1
2
3
4
5
Control
1
2
3
4
5
Control
1
2
3
4
5
Medium A
Control
0
0
Medium C
Case Study 2
Summary and Conclusions
!  Production levels can vary depending on the base medium and
supplementation combination – even with the same cell line.
!  CD supplement driven production can vary between cell lines. Some
of this is dictated by cell line genetics.
!  Screening of basal media in combination with supplement screening
will yield the most optimal combinations for success.
Case Study 3
Optimize Feed Strategy For Optimal Performance
Experimental setup:
CHO cell line expressing mAb
Two CD supplements
Three feed strategies
A feed strategy optimal for one supplement may not be optimal for another.
Case Study 3
Summary and Conclusions
•  Type of supplementation and supplementation
strategies affect growth and production performance.
•  A feed strategy optimal for one supplement may not
be optimal for another.
Case Study 4
Bioreactor Optimization:
Medium and Nutritional Feeds
Experimental setup:
CHO cell line expressing mAb
Cultures are performed in basal media selected
as optimal in shaker flasks batch culture
Scale to 1L bioreactor cultures
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Bioreactor parameter optimization was not sufficient to increase culture longevity.
Spent medium analysis showed lactate accumulation and amino acid consumption.
Medium feed on Day 3 of culture lowered lactate levels and slightly enhanced culture
longevity, VCD, and viability.
Nutritional feed was included at Days 0 and 3 to further optimize the basal process.
This modification significantly improved culture longevity, VCD and viability.
Case Study 4
Bioreactor Optimization:
Medium and Nutritional Feeds
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Addition of nutritional feed shifted lactate to non-toxic levels, possibly by promoting a shift to
lactate consumption.
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Production was not significantly enhanced by addition of nutritional feed compared to control
medium. This correlated to shaker flasks.
Case Study 4
Bioreactor Optimization:
CD Supplement optimization
Different feed strategies can show different growth, metabolic and production profiles
Case Study 4
Bioreactor Optimization
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Optimized process: FB4 with CD2 supplement feed on Day 3 and Day 5
CD supplement 2 increased protein production approx. 2.5 fold over the optimized base process.
Case Study 4
Summary and Conclusions
!  Each combination of cell line, base medium, and supplementation
requires process optimization.
!  Process optimization includes bioreactor operational conditions but may
require further nutritional optimization.
!  Control of toxic metabolite levels, through various strategies, is imperative
for optimized culture performance.
!  Due to improved mass transfer and controlled conditions, nutrient
consumption can be different in bioreactor culture compared to shaker
flask culture.
!  Only through proper base line process optimization can the value of CD
supplements be unveiled.
Technical
Summary and Conclusions
!  CHO cell lines are highly genetically divergent which can lead to divergent cell
culture performance.
!  Basal media and supplementation performance are interdependent and cell line
specific.
!  Optimization of basal media is critical to CD supplementation.
!  CD supplement performance may vary across CHO cell lines under differing
culture conditions.
!  Thorough process optimization may be required to reap the benefits of CD
supplementation.
!  Shaker flasks, ambr™ microbioreactor, 1L bioreactors
BD Resurge™ CD Supplements
!  Five (5) BD Resurge™ CD Supplements.
!  The supplements are fully chemically defined (CD) and animal free (AF).
!  These supplements have been developed on a variety of host CHO cell lines
and in various culture systems including shaker flasks and bench-scale
bioreactors.
!  BD Resurge™ CD Supplements are available as a kit: BD Resurge™ CD PAK.
!  1 of each of the five BD Resurge™ CD supplements @ 100 gram bottles
!  BD Resurge™ CD Supplements are available as individual pack outs.
!  100 grams, 1Kg, 5Kg
BD Resurge™ CD Supplements
!  BD Resurge™ CD PAK offers a diverse panel of options for
increasing bioproduction cell culture performance.
!  Performance of BD Resurge™ CD supplements are competitive
against commercially available CD supplements.
!  BD Resurge™ CD supplements are scalable from shaking deep wells
to bioreactors.
!  BD Resurge™ CD supplements can be used in both batch and fed
batch applications.
!  Media, cell line and feeding strategies are critical factors for the
successful evaluation of CD supplements.
BD Resurge™ CD Supplements
PRODUCT NAME
BD Resurge™ CD1
BD Resurge™ CD2
BD Resurge™ CD3
BD Resurge™ CD4
BD Resurge™ CD5
BD Resurge™ CD PAK
BD CATALOG #
QUANTITY
670011
100G
670012
1KG
670013
5KG
670015
100G
670016
1KG
670017
5KG
670018
100G
670019
1KG
670020
5KG
670021
100G
670022
1KG
670023
5KG
670024
100G
670025
1KG
670026
5KG
670030
5 X 100G
Acknowledgements
BD Biosciences Advanced
Bioprocessing
Product Development Teams!
Trademarks are the property of their respective owners. BD, BD Logo, and BD Resurge are trademarks of Becton, Dickinson and Company. © 2013 BD
Send ques)ons to: [email protected]

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