© 2009 SAFC Business confidential
Defining Hydrolysates
An Approach for Generating a Chemically Defined Alternative
Zachary DeedsSenior R&D [email protected] 18th, 2009
© 2009 SAFC Business confidential
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Overview
• Background
• Our Approach
• Fraction Identification
• Microarray Data
• Product Information
• New Commercial Proofs
Chemically Defined, Animal Component Free, & Synthetic!
© 2009 SAFC Business confidential
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What are Hydrolysates?
The protein sources for the more commonly used hydrolysates are soy, wheat gluten, meat and yeast*.
http://www.sheffield-products.com/pharma_ingredients/ http://www.dmv-international.com
© 2009 SAFC Business confidential
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Possible Hydrolysate Functions
• Nutritive
– Free amino acids/small peptides
– Carryover of carbohydrates and vitamins
• Protective effect
– Antiapoptotic activity has been reported
– Sheer force
• Cell stimulation
– Peptides can mimic growth/survival factors
• The benefits may be obvious, but the cellular and molecular mechanisms of action are poorly understood
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Drivers for Alternatives
• Risk Mitigation
– Undefined nature
– Lot-to-lot variability
– Regulatory pressure
• Challenges
– Extraordinarily complex components; major simplification is needed
– Analytical expertise and equipment
– Time investment
© 2009 SAFC Business confidential
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Our General Approach• Goals
– Provide a solution that mitigate risks while maintaining performance
• Leverage SAFC capabilities to define the key components of hydrolysates
• Part 1
– Develop “Base” supplement (free AA, metals, vitamins)
• Part 2
– Use RP-HPLC to fractionate multiple hydrolysates
– Identify bioactive fractions with CHO cells
– Use analytical techniques to identify components in the fractions
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FlowchartScreen Hydrolysates
RP-HPLC Fractionate & Lyophilize
Screen Fractions (+ Base) with CHO
ID Fraction Components & Source CD Versions
Combine and Optimize with Base Supplement for
Final Product
Screen Components with Multiple CHO Lines
EX-CELL® CD Hydrolysate
Fusion
© 2009 SAFC Business confidential
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Method for Fractionation
• Followed the poster for “Peptide Mapping of a Wheat Gluten Hydrolysate by Reversed Phase High Performance Liquid Chromatography (RP-HPLC)”
– C-18 column (25 cm x 2.12 cm) – 10 µm particle size
– Mobile phase A 0.1% TFA in water
– Mobile phase B 0.1% TFA in acetonitrile
– 300 min. linear gradient from 0-30% B (hold 30 min at 30%)
– Flow rate 5 mL/min
– Collected data at 280 nm
– Injection volume was 5 mL of 200 g/L hydrolysate stock
– Collected fractions in 5-minute intervals
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RP-HPLC Fractionation (Reproducibility)
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Fraction Screening Protocol• Cell Line: SAFC Biosciences CHO cell line that produces a
recombinant h-IgG
• Medium: SAFC Biosciences formulation with hydrolysates removed
– Note: Cell line must show a significant response to hydrolysate feeding in the chosen medium
• Cell culture assay was run in duplicate in TPP bioreactor PVC tubes
• Cultures inoculated at 2e5vc/mL & fed at 8e5-1.2e6vc/mL (Day 2)
• Fraction Feeding:
– Glucose
– “Nutritive” Base Supplement (AA, vitamins, metals/salts)
– Fractions at relative 2 g/L level
© 2009 SAFC Business confidential
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Fraction Screening - Growth
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
9.00E+06
0 1 2 3 4 5 6 7 8
Day
Via
ble
Ce
lls/m
L
No Feed
Base Supplement
Yeast Extract 2g/L
Base + Fraction A
Base + Fraction B
Base + Fraction C
Base + Fraction D
Feed
© 2009 SAFC Business confidential
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Fraction Screening - IgG Productivity
*Early Productivity Stimulation
-40.0%
-20.0%
0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
No
Fe
ed
Ba
se
Su
pp
lem
en
t
Ye
as
tE
xtr
ac
t 2
g/L
Fra
cti
on
A
Fra
cti
on
B
Fra
cti
on
C
Fra
cti
on
D
% C
han
ge
fro
m B
ase
Co
ntr
ol
4
7
Day:
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Fraction ComplexitySize Exclusion Chromatography:
Standards
Hydrolysate
Fraction
1651183 362
6511
3496
66,400
12,327
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Analytical Identification
• Significant time was spent working on secondary separation methods
• Multiple analytical techniques were used to analyze the fractions
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Positive Result Example
• A compound was identified in several positive fractions from different hydrolysates
• Upon screening, a large positive impact was seen
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Example - Growth
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
9.00E+06
1.00E+07
0 1 2 3 4 5 6 7 8
Day
Via
ble
Ce
lls/m
L
Hydrolysate 2g/L
Base Supplement
Base + Compound
Feed
© 2009 SAFC Business confidential
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Example – IgG Production
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
Hy
dro
lys
ate
2g
/L
Ba
se
Su
pp
lem
en
t
Ba
se
+C
om
po
un
d% C
han
ge
fro
m B
ase
Co
ntr
ol
5
7
Day:
© 2009 SAFC Business confidential
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Positive Result Example
• Titration of the compound was performed and it was added to make a new “Base” supplement
• Further optimization of the “Base” was necessary to address other component limitations
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Microarray Data
• In addition to traditional optimization methods, our group also utilizes additional advanced techniques to seek out ways to improve cell culture performance
• Microarray analysis is one of these tools and it was applied to study the effects of hydrolysates
© 2009 SAFC Business confidential
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Experimental Design• SAFC Biosciences CHO cell line that produces a recombinant h-IgG
• Base media formulation is CD (Chemically Defined)
• Cultures fed at a VCD of 8e5 - 1.2e6 cells/mL (Day 2)– No feed– Glucose only feed– + Soy hydrolysate feed– + Yeast hydrolysate feed– + EX-CELL CD Hydrolysate Fusion (CD Feed)
• Cell culture assay was run with biological triplicates in TPP bioreactor PVC tubes.
• Samples collected for RNA on D2, D3, D5, D7 and D10
• Microarray analysis was done with biological and technical duplicates.– Dye swaps– Reference pool comparison– Glucose only feed as the control
© 2009 SAFC Business confidential
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CHO Microarray Platform
• SAFC CHO probes >30,000
• Mouse orthologous probes ~10,000
• SAFC control probes 168
• Agilent control probes 1,400
• Total # of features ~44,000
44K
4 X 44K custom CHO Array
Agilent QC Analysis
Raw Data Processing
Statistical Analysis Using
Genesifter
Gene Expression and Pathway Analysis Using Genesifter and Ingenuity
CHO Sequence Database
~60,000 total sequences
~20,000 unique sequences
~9,000 annotated contigs
© 2009 SAFC Business confidential
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Hydrolysates Increase VCD and qP Average Viable Cell Density
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
5.0E+06
6.0E+06
7.0E+06
8.0E+06
9.0E+06
0 1 2 3 4 5 6 7 8 9 10 11
Days in Culture
VC
D (
cell
s/m
l)
No Feed Glucose Soy Yeast CD Feed
Average Productivity
0
100
200
300
400
500
600
700
Day 4 Day 5 Day 7 Day 10
IgG
(u
g/m
l)
No Feed Glucose Soy Yeast CD Feed
Average Specific Productivity
0
5
10
15
20
25
30
35
40
45
50
No Feed Glucose Soy Yeast CD Feed
qP
(p
g/c
ell/d
ay)
© 2009 SAFC Business confidential
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Unique to Soy
33 34
Unique to Yeast
279 73
Common 222 78
Up Down
Differentially Expressed Genes in Response to Hydrolysate Feeds
Adjusted p-value<0.05 1.4 Fold Change
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CD Hydrolysate Fusion Comparison
Adjusted p-value<0.05 1.4 Fold Change
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Molecular and Cellular Function Analysis of Differentially Expressed Genes
Adjusted p-value<0.05 1.4 Fold Change
© 2009 SAFC Business confidential
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Molecular and Cellular Function Analysis of Differentially Expressed Genes
Adjusted p-value<0.05 1.4 Fold Change
© 2009 SAFC Business confidential
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New Product Development
• EX-CELL CD Hydrolysate Fusion:
– Ready to use 20X liquid format (14700C)
– Proven powder equivalency (24700C)
– Designed for CHO, but has applications with NS0, SP2/0, and others
– Manufactured specifically for Industrial Cell Culture applications
• Chemically Defined (no fractions)
• Animal Component Free
• Synthetic Components Only
© 2009 SAFC Business confidential
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Scalability & Stability
• Production Concerns
– Solubility, OSMO, pH
– Scalability
– Supply Chain (ACF)
• Product Stability
– Significant time was invested in generating a concentrated, stable product
– Accelerated stability testing of the liquid at two temperatures (37°C and RT) has predicted at least 18 month to 24 month shelf life
© 2009 SAFC Business confidential
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Product Performance – Batch
• Example
– Batch Mode with an IgG producing CHO clone
• Grown in a proprietary SAFC Biosciences hydrolysate-containing medium that was optimized for this cell line
• A hydrolysate-free batch was made by imMEDIAte ADVANTAGE
© 2009 SAFC Business confidential
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Batch Substitution
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
5.0E+06
6.0E+06
7.0E+06
8.0E+06
0 1 2 3 4 5 6 7 8 9 10 11
Day
Via
ble
Ce
lls/m
L
No Hydrolysate
HydrolysateControl
CD Hydrol. Fusion1x
© 2009 SAFC Business confidential
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Batch Substitution
0
100
200
300
400
500
Max IgG
mg
/L I
gG
No Hydrolysate
Hydrolysate Control
CD Hydrol. Fusion 1x
© 2009 SAFC Business confidential
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Product Performance – Fed-Batch
• Example
– Fed-Batch with an IgG producing CHO clone (GS)
• Grown in a new SAFC Biosciences chemically defined medium (EX-CELL CD CHO Fusion, 14365C)
– Fed on Day 2 with 6 g/L glucose and respective feed
© 2009 SAFC Business confidential
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Fed-Batch Substitution
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
3.0E+06
3.5E+06
4.0E+06
4.5E+06
5.0E+06
0 1 2 3 4 5 6 7 8 9 10 11
Day
Via
ble
Ce
lls/m
L
Glucose Only Soy 2g/L Soy 4g/L
Yeast 2g/L Yeast 4g/L CD Hydrolysate Fusion 1x
Feed
© 2009 SAFC Business confidential
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Fed-Batch Substitution
0100200300400500600700800900
10001100120013001400
Glu
co
se
On
ly
So
y U
F 2
g/L
So
y U
F 4
g/L
Ye
as
tE
xtr
ac
t 2
g/L
Ye
as
tE
xtr
ac
t 4
g/L
CD
Hy
dro
lys
ate
Fu
sio
n 1
x
Max
. m
g/L
Ig
G
© 2009 SAFC Business confidential
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Quality ProfileCEX Product Quality Analysis
0
5
10
15
20
25
30
35
40
45
50
Acidic Peak 1 Peak 2 Peak 3 Basic
% o
f P
eak
Are
a
Hydrolysate Process
CD Hydrolysate FusionProcess
Product Quality Analysis of IgG produced by CHO-IgG1. A CEX-HPLC method was used to look at product quality difference between IgG produced by a process using hydrolysates and the CD Hydrolysate Fusion.
© 2009 SAFC Business confidential
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New Product Summary• Product Expectations
– In our in-house studies, EX-CELL CD Hydrolysate Fusion can recover 80% to >100% of the performance of hydrolysates in a basal medium or a fed-batch process
– Responses can vary depending on both the cell line and basal medium
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By the Numbers
• 1 Million plus liters of base media that incorporates CD Hydrolysate Fusion forecasted in 2010
• 1000 plus liters of base media that incorporates CD Hydrolysate Fusion required for vaccine manufacturer validation in Q1 2010
• 9 plus active therapeutic process evaluations
• 2 forecasted future commercial therapeutic process incorporations
All in only 4 months since Launch
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