Optimized Feed Strategies for Increased Titer and … world leader in serving science Steve Gorfien, PhD Optimized Feed Strategies for Increased Titer and Product Quality Senior Director,

The world leader in serving science

Steve Gorfien, PhD

Optimized Feed Strategies for Increased Titer and Product Quality

Senior Director, R&D

[email protected]

2

Agenda

Pain points in fed-batch culture

Addressing pain points

• Nutritional and dilutional gaps

• Process considerations

• Importance of product quality

• Factors that can influence product quality

• Latest advance in high-throughput glycan testing

Conclusions

3

Pain Points in Fed-Batch Culture

Nutritional gap

• Engineered cells have high nutritional demands

• Providing the right nutrients at the right time

• Matching basal medium and feed

Dilutional gap

• Dilution impact of feed addition

• How concentrated can you make the feed to be able to feed more?

Process

• High/low pH feeds add to preparation complexity and time

• Pick clones in a system that reflects ultimate process

Desired end point

• Cell density, product titer, product quality

• High cell density may not correlate with high titer or with desired critical quality attributes

4

Engineered Cells Have High Nutritional Demands

0

50

100

150

200

250

300

350

400

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11

EP

O t

ite

r (m

g/L

)

Via

ble

ce

ll d

en

sit

y (

10

6 c

ell

s/m

L)

Run time (days)

0 2 4 6 8 10 12

Co

nc

en

tra

tio

n

Run time (days)

Arginine Asparagine Cysteine Glutamine

5

Balanced Formulations Drive Performance

Sample target process

Perform spent media analysis Analyze growth/titer

Calculate consumption rates

Analyze nutritional pathways Optimized balanced formulations

Via

ble

cell

density (

VC

/mL)

IgG

titer

(mg/L

)

Time (days)

6

Benefit of Rationally Integrated Optimization

Cell Line/control process

• CHO-K1SV–high IgG expression

• Modified catalog base medium

• Proprietary feed/process

Desired outcome

• Improved titer

• Proprietary base medium

• Formulation access

• Stay with current feed/process

Modification of basal medium alone was insufficient to improve titer

Jiang et al, BioProcess International 10(3) March, 2012

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1 2 3 4 5

Re

lati

ve

Ig

G t

ite

r

Steps

7

Process Intensification Addresses Nutritional and Dilutional Gaps

Higher total productivity by maintaining qp

Pro

tein

tit

er

Integral viable cell density

Theoretical trend

Dilutional gap

Nutritional gap

Nutrient feeds

Glucose feed only

8

IVC (1012 viable cell days)

IgG

tit

er

(g)

0 0.002 0.004 0.006 0.008 0.01

0.05

0.1

0.15

0.2

0.25

0.3

0

qp= 29 pg/cell/day

CD OptiCHO

CD FortiCHO

CD OptiCHO w/ EFC

CD FortiCHO w/ EFC

Sustaining qP Increased QP in Matched Basal/Feed Media

Elimination of nutritional gap = ~2-fold titer increase

Via

ble

ce

ll d

en

sit

y (

10

6 c

ell

s/m

L)

Culture time (days)

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14 16

IgG

tit

er

(mg

/L)

0

500

1000

1500

2000

2500

3000

CD OptiCHO

CD FortiCHO

CD OptiCHO w/ EFC

CD FortiCHO w/ EFC

9

Granulated Format Improves Process and Productivity

Processing time cut in half vs. DPM

Product

Solution

delivery

Spray nozzle

Air preparation unit

Neutral pH pH range: 6.5–7.1

Super-concentrated feeds Target: 150–200 g/L

Simple to prepare Just add water

Faster hydrating ≤90 min

Flexible in its use Add less water to concentrate

Fluid granulation

Solidifying

Solid bridge

Moistening

Liquid bridge AGT granule

Finished granule Spraying

Trace liquids Powder

10

Increased Titer with Highly Concentrated Feeds

30–40% titer increase by eliminating the dilutional gap

0

20

40

60

80

100

120

Traditional Dilution Maximumworking volume

Available space

Feeding supplement

Media working volume

~20% improvement in

bioreactor use

3x

3x

71 L

29 L 19 L

10 L

71 L 88 L

12 L

mAb A, EFC (45%)

mAb B, EFC (45%)

mAb A, 1X EFC+ (45%)

mAb B, 1X EFC+ (45%)

mAb A, 2X EFC+ (22.5%)

mAb B, 2X EFC+ (22.5%)

Culture time (days)

6 8 10 12 14 16 0

500

1000

1500

2000

2500

3000

An

tib

od

y t

ite

r (m

g/L

)

Vo

lum

e (

L)

11

Clone Ranking Varies with Process

0

200

400

600

800

1000

1200

1400

1600

1800

34F05 54A05 02B07 05B09 05D09 25F10 05C07 60D06 55F02 05A03

Simple Fed-batch

Fed-batch 1

Fed-batch 2

Top 10 MabB Clones in SFB & FB

Clone ID

Tit

er

(mg

/L)

12

Product Quality is Important

Critical quality attribute:

“A physical, chemical, biological or microbiological property or characteristic that should be within an appropriate

limit, range or distribution to ensure the desired product quality”*

* ICH, ICH Harmonized Tripartite Guideline Q8: Pharmaceutical Development, Step 4 version (August 2009)

Examples of critical quality attributes of biotherapeutics:

• Glycosylation

• Charge variants

• N- and C- terminal structural features

• Fragmentation

• Aggregation

13

Glycosylation is a Key Critical Quality Attribute

Glycosylation can affect:

• Protein folding

• Solubility

• Immunogenicity

• Enzymatic activity

• Transport

• Turnover rate

Activity and availability of the protein are impacted by glycans

NA2G1F

NA2FA2F

A1F

NGA2F

A1

NA2G1

Man-9

Man-8

Man-7

Man-5

Man-6

NA2G1F

NA2FA2F

A1F

NGA2F

A1

NA2G1

Man-9

Man-8

Man-7

Man-5

Man-6

14

Productivity Increase While Maintaining Quality Profile

No change in key glycan structures

0%

10%

20%

30%

40%

50%

60%

70%

80%

CD OptiCHO medium + 30% EFA CD OptiCHO medium + 30% EFA

+ 10% FMax

Percent glycosylation at harvest

G0F G1F G2F G0 M5

CD OptiCHO medium

+ 30% EFA

0 50 100 150 200 250

0

50%

100%

150%

200%

250%

Pro

tein

tit

er

Integral viable cell density

0

50%

100%

150%

200%

250%

Pro

tein

tit

er

Culture duration (days)

EFA = Gibco™ CHO CD EfficientFeed™ A Supplement and Fmax = Gibco™ FunctionMAX™ Titer Enhancer

CD OptiCHO medium

+30% EFA

+10% FMax

15

Addressing Media and Process Impact on Glycosylation*

• pH

• DO, pCO2

• Temperature

• Low glucose/glutamine

• Mn, NH3, glycerol, nucleotide sugar content

• Cell viability at harvest

• Shear stress

• Perfusion vs. fed batch

* From review by Hossler P, Khattak SF and Li KJ, Glycobiology, 19(9):936-949, 2009

16

Preventing Ammonia Accumulation Galactosylation

0

10

20

30

40

50

60

70

80

90

100

Condition 1 Condition 2 Condition 3

G0F

G1F

G2F

G0

M5

Day 14: NH4+: 9 mM Day 14: NH4+: 6 mM Day 14: NH4+: 4.5 mM

Pe

rce

nt

(%)

17

Controlled Conditions in Bioreactors Galactosylation

0

10

20

30

40

50

60

70

80

90

Bioreactor Shake flask

G0F

G1F

G2F

G0

G1

M5

Pe

rce

nt

(%)

18

Lower Harvest Viability G0F, G1F

0

10

20

30

40

50

60

70

80

90

Day 14 Day 19

G0F

G1F

G2F

G0

G1

M5

Pe

rce

nt

(%)

95% Viability 45% Viability

Harvest day

19

CHO Transcriptome Comparison

Glycosylation differences between cell lines linked with transcriptome profiles

Increased transcripts in DG44: ALG2, ALG3, ALG9 and ALG12, ALG8 and ALG10, ALG14, and B4GALT5

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Day 5 Day 7 Day 11 Day 14

G0F

G1F

G2F

G0

G1

M5

CHO-S

0%

10%

20%

30%

40%

50%

60%

70%

80%

Day 5 Day 7 Day 11 Day 14

G0F

G1F

G2F

G0

G1

M5

DG44 CHO

Higher terminal galactose in DG44

20

Media/Feed Composition Influences Glycosylation

0

10

20

30

40

50

60

70

80

90

G0F

G1F

G2F

G0

G1

M5

Pe

rce

nt

(%)

21

Rational Glycan Modulation

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Hu

nd

red

s

G0F

G1F

G2F

G0

M5

% G

lyc

osyla

tio

n

22

Additives Only Provided Partial Modulation of Glycans

An additive approach alone did not provide the ability to target a desired glycan profile

0

10

20

30

40

50

60

70

80

90

100

A1F/MAN5 G0F G1F_1 G1F_2 G2F

control

0.03X

0.17X

0.36X

0.68X

0.87X

1.00X

% R

ela

tive

qu

an

tity

23

Another Approach Was Tried Using Gibco™ GlycanTune™ C+ Total Feed

0

2

4

6

8

10

12

14

16

18

VC

D (

x1

06 c

ell

s/m

l)

0 2 4 6 8 10 12 14 16

Culture time (days)

A

Glucose control

20% 2X EFC+ Control

15% 3X GTC+ Transition day 15







B

4 6 8 10 12 14 16 0

20%

40%

60%

80%

100%

120%

Culture time (days)

IgG

tit

er

(% o

f c

on

tro

l)

24

Another Approach Was Tried Using GlycanTune C+ Total Feed

The timing of transition from Gibco™ EfficientFeed™ C+ (EFC+) to GlycanTune C+ (GTC+)

makes it possible to target specific glycosylation profiles.

0

10

20

30

40

50

60

70

80

EFC+Control

Day 15 Day 13 Day 11 Day 9 Day 7 Day 5 Day 4

G0F

G1F_1

G1F_2

G2F

Mann5/A1F

Day of transition from EFC+ to GTC+

% R

ela

tive

qu

an

tity

25

Results Solutions

Case Study: Modulating Glycan Profile

Problem

Starting point:

Customer was satisfied with yield

but required product quality

modulation to match the innovator

molecule (4/12 glycan attributes

within an acceptable range) In-house

program

Analyze

product

quality data

DoE using

in-house

IP/literature

Evaluate in

ambr 15 HT

system

Client

analyzes PQ

Optimization

SA2

SA2F

SA1

SA1F

M5

G0

G0F

G1

G1Fa

G1Fb

G2

G2f

PQ improvement

Outcome: 9 of 12 attributes within range,

remaining 3 within 80% of target.

26

Current Glycan Analysis Solutions

• Single channel (CE & LC) low-throughput (days/96 samples) separation

• Poor data quality of high-throughput methods

• Labor intensive workflow (pipetting, centrifugation, etc.)

• Use of toxic sodium cyanoborohydride chemistry

• Use of vacuum centrifugation steps

• Generic software taking long analysis time

• Non integrated solution from multiple vendors

• Commercial kits with high cost per sample

• No validation support for result/solution

Kit supplier Instrument

supplier

Generic

software

Non integrated

solution

27

Key Features Desired for a High-Throughput Glycan Analysis Platform

Easy sample prep

• Magnetic bead-based sample prep

• Hands-on time <3 hrs for 96 samples

• Fewer pipetting steps

• No sodium cyanoborohydride use

• No vacuum centrifugation steps

High throughput

• Sample prep and data of 96 samples in 7–9 hrs

Resolution

• Sialylated glycans

• Structural isomers

• Fucose species

• High-mannose species

Dye labeling

• Multiple dyes with superior sensitivity

High sensitivity

• Analyze as little as 1 µg of IgG

Low cost

• Robust instrument and capillaries with low running cost

Fully integrated

• Full sample prep, hardware, and software solution

28

Data analysis

Fit for use software

Robust alignment

algorithms

Data collection

Multi-capillary

Run-to-run reproducibility

Cap-to-cap concordance

Dye labeling

Multiple dyes

Reactivity

Sensitivity

Resolution

No dry down

Glycan purification

Reproducible & quantitative

(Magnetic beads)

Novel CE-based HT Glycan Analysis System

Glycan release

Fast <1 hr IgG inputs

(1.6–50 µg)

Complete sample prep and data of 96 samples in 7–9 hours

29

Different Dyes for Better Specificity

Separation of major glycans was possible

APTS

Turquoise™

Teal™

30

Conclusions

• Improved volumetric productivity (Qp) can be achieved by use of rationally

designed, matched basal and feed media to sustain specific productivity (qp)

• A granulated feed format enables highly concentrated feeds which can

further improve productivity and ease of use

• Product quality can be impacted by choice of media, feed, and

process conditions

• High-throughput glycan analysis by capillary electrophoresis simplifies and

improves the workflow

Future challenges:

• Methods for custom optimization of other critical quality attributes, including

charge variant and aggregation profiles

• Rapid, high-throughput, and sensitive methods for analysis of other

critical quality attributes

31

Acknowledgments

• Ryan Boniface

• Michael Gillmeister

• Jaime Goldfuss

• Shawn Barrett

• Mark Stramaglia

• Serena Smith

• Doan Tran

• Baburaj Kunnummal

• Shaheer Khan

• JenKuei Liu


Caution: For use as a raw material in further manufacturing applications

© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

For more information on glycosylation, visit thermofisher.com/GlycanTune

https://www.thermofisher.com/order/catalog/product/A2971901?ICID=search-product




Appendix

34

Another Approach Was Tried Using Gibco™ GlycanTune™ A+ Total Feed

B

18

A

0

2

4

6

8

10

12

14

16

VC

D (

x1

06 c

ells

/mL

)

0 2 4 6 8 10 12 14 16

Culture time (days)

18

Culture time (days)

0

20%

40%

60%

80%

100%

120%

IgG

tit

er

(% o

f c

on

tro

l)

0 2 4 6 8 10 12 14 16

Glucose control

20% 2X EFC+ control

15% 3X GTC+ transition day 15







A. The growth profiles for the DG44 cell in Gibco™ CD OptiCHO™ medium were similar

with both EFA+ and with the use of or when transitioning to GTA+.

B. DG44 titer comparison between feeding conditions. 100% titer is the titer of 15% 3X

EFA+ on day 16. Titer results indicate that the use of and transition to GTA+ does not

negatively affect protein production.

0

10

20

30

40

50

60

70

80

EFA+Control


G0F

G1F_1

G1F_2

G2F

Mann5/A1F

Day of transition from EFA+ to GTA+

% R

ela

tiv

e q

uan

tity

The timing of transition from Gibco™ EfficientFeed™ A+ (EFA+) to GlycanTune A+ (GTA+) makes

it possible to target specific glycosylation profiles. Modulating G0F from 72% down to 30%,

while increasing G1F (1 and 2) and increasing G2F.

35

Another Approach Tried with Gibco™ GlycanTune™ B+ Total Feed

Also works with other matched basal/feed media

Culture time (days)

Culture time (days)

B

A

A. The growth profiles for the DG44 cell in CD CHO medium were similar with both EFB+ and when transitioning

to GTB+. The large error bar at day 10 was due to clumped cells.

B. DG44 titer comparison between feeding conditions. 100% titer is the titer of 15% 3X EFB+ on day 16. Titer

results indicate that the use of and transition to GTB+ does not negatively affect protein production.

Glucose control

20% 2X EFC+ control








0 2 4 6 8 10 12 14 16

4 6 8 10 12 14 16

IgG

tit

er

(% o

f c

on

tro

l)

VC

D (

x1

06 c

ells

/mL

)

0

2

4

6

8

10

12

14

16

18

0

20%

40%

60%

80%

100%

120%

0

10

20

30

40

50

60

70

80

90

EFA+Control


G0F

G1F_1

G1F_2

G2F

Mann5/A1F

Day of transition from EFA+ to GTA+

% R

ela

tiv

e q

uan

tity

The timing of transition from Gibco™ EfficientFeed™ B+ (EFB+) to GlycanTune B+ (GTB+) makes it

possible to target specific glycosylation profiles. Modulating G0F from 80% down to 41%, while

increasing G1F (1 and 2) and increasing G2F.

Documents

Optimized Feed Strategies for Increased Titer and … world leader in serving science Steve Gorfien, PhD Optimized Feed Strategies for Increased Titer and Product Quality Senior Director,