hydrolysate characterization technical presentation webinar11 2009

© 2009 SAFC Business confidential

Defining Hydrolysates

An Approach for Generating a Chemically Defined Alternative

Zachary DeedsSenior R&D [email protected] 18th, 2009


2

Overview

• Background

• Our Approach

• Fraction Identification

• Microarray Data

• Product Information

• New Commercial Proofs

Chemically Defined, Animal Component Free, & Synthetic!


3

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


4

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


5

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


6

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


7

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


8

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


9

RP-HPLC Fractionation (Reproducibility)


10

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


11

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


12

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:


13

Fraction ComplexitySize Exclusion Chromatography:

Standards

Hydrolysate

Fraction

1651183 362

6511

3496

66,400

12,327


14

Analytical Identification

• Significant time was spent working on secondary separation methods

• Multiple analytical techniques were used to analyze the fractions


15

Positive Result Example

• A compound was identified in several positive fractions from different hydrolysates

• Upon screening, a large positive impact was seen


16

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


17

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:


18

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


19

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


20

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


21

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


22

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)


Average Specific Productivity

0

5

10

15

20

25

30

35

40

45

50


qP

(p

g/c

ell/d

ay)


23

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


24

CD Hydrolysate Fusion Comparison



25

Molecular and Cellular Function Analysis of Differentially Expressed Genes



26

Molecular and Cellular Function Analysis of Differentially Expressed Genes



27

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


28

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


29

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


30

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


31

Batch Substitution

0

100

200

300

400

500

Max IgG

mg

/L I

gG

No Hydrolysate

Hydrolysate Control

CD Hydrol. Fusion 1x


32

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


33

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


34

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


35

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.


36

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


37

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


38

hydrolysate characterization technical presentation webinar11 2009

Documents

soy hydrolysate feed

yeast hydrolysate feed

standards hydrolysate

multiple hydrolysates

positive fractions

feed glucose soy

cho cells

screen fractions