Building Lower Cost Upstream Vaccine Processes · Designed for vaccine production in Vero cells Adherent/Microcarrier Designed for vaccine production in BHK-21 cells Suspension Designed

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Building Lower Cost Upstream Vaccine Processes Abhijeet Kohli, Angel Varela 30-Nov-2016

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Agenda for today’s discussion

Characteristics of a low cost process

Cell Substrate Selection

Cell Culture Media

Selection of Production Scale

Conclusions

Proprietary & Confidential

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Low cost does not mean low price

Low Cost Process Lower Priced

Components


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There are several ways to assess the cost of a process


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Cost savings go hand-in-hand with process efficiency

0%

20%

40%

60%

80%

100%

120%

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600

0% 100% 200% 300% 400%

Co

st p

er

Do

se

Sim

ula

ted

Do

ses

pe

r B

atch

Simulated Productivity Improvement

Doses Cost per Dose

100% Productivity Improvement = 0.3 log10TCID50/mL Titer Increase

Making more doses with equivalent or increased batch cost could still lead to an overall reduction in

cost/dose

Assuming dose improvements occur at constant cost



Cell Substrate Selection


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Importance of Cell Substrate Selection

Imposes Constraints

• Adherent vs. Suspension

• Primary vs. Cell-line

• Serum vs. Serum-Free

Could Increase Necessary Investment

• Licensing Costs

• Cell Banking Costs


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Cell substrate choice is an important first step

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Vero COS-7 MRC-5 C6 MDCK RK13 CEF PK(15) 293T HeLa BHK-21 WISH CHO-K1

Vac

cin

ia T

ian

Tia

n F

old

Incr

eas

e

MRC-5 (Adherent Primary) and CEF (Adherent Primary) have been used in the past

~3x higher titer achieved using Vero Cells (Adherent Cell-line)

Fang et al. 2005 Virology. 335


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Cell substrate choice is an important first step

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Log 1

0CC

ID5

0/m

L

POLIO1 POLIO2 POLIO3

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1

2

3

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7

8

9

Lo

g10C

CID

50/m

L

RSV

0123456789

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Log 1

0CC

ID5

0/m

L

H1N1 H3N2

Vlecken et al. 2013 Journal of Virological Methods. 193

Compromise in productivity may be necessary to

• Select substrates that can grow multiple virus serotypes

adequately

• Allow suspension culture without microcarriers

• Have a commercial serum free medium available


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• Knocking out inhibitory genes could yield higher virus titers.

• Long and intensive screening process

Cell Line Engineering Could Enhance Titer Further

Screen & Verify

Gene Targets

Identify Titer

Enhancement

Test in Combination

to Assess Synergy


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Cells may be further modified to gain higher yields

Control

Sanden et al. 2016 Journal of Virology. 90(4)

Gene knockdown performed with siRNA in Vero cells

~80x improvement in Polio virus titer


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Cells may be further modified to gain higher yields

Control

Sanden et al. 2016 Journal of Virology. 90(4)

Same genes also improve EV71 titer

A license must be acquired from Thermo Fisher to use

these gene targets to commercialize vaccine processes



Cell Culture Media


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Customers Typically Compare Price of Basal Media C

lassic

al M

edia

S

eru

m-F

ree M

edia

Basal Medium

Serum Free

Medium

* US catalog pricing based on 100L of media

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20

40

60

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Classical SFM

Ave

rage

pri

ce p

er

L

Base Medium


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Serum Free Media Appears More Expensive by Comparison C

lassic

al M

edia

S

eru

m-F

ree M

edia

Basal Medium Antibiotic

Serum Free

Medium Antibiotic L-glutamine


L-glutamine

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120

Classical SFM

Ave

rage

pri

ce p

er

L

Base Medium Antibiotic L-Glutamine


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Inclusion of the Cost of FBS Changes the Equation C

lassic

al M

edia

S

eru

m-F

ree M

edia


Serum Free



L-glutamine FBS

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120

Classical SFM

Ave

rage

pri

ce p

er

L

Base Medium Antibiotic L-Glutamine FBS

Serum containing medium is more expensive than

serum free media


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Inclusion of the Cost of FBS Changes the Equation C

lassic

al M

edia

S

eru

m-F

ree M

edia


Serum Free



L-glutamine FBS

0

20

40

60

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120

Classical SFM

Ave

rage

pri

ce p

er

L


Serum containing medium is more expensive than

serum free media


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Final Average Cost Lower Because FBS Not Used in Production C

lassic

al M

edia

S

eru

m-F

ree M

edia


Serum Free



L-glutamine FBS

Assuming 50% of medium used is for Growth and

50% for production

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40

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Classical SFM

Ave

rage

pri

ce p

er

L



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Added Benefits to Serum Free Media

Cell Factories

Cell

Growth

Cell Factory

Plant

Virus Seed

Wash with

Buffer Bulk Harvest

Transfer

Growth Medium

10% FBS Wash Production Medium

0% FBS

Why?

• WHO requires that no more than 50ng of BSA may be present in a vaccine dose

• Residual BSA from serum can negatively impact this specification

• Serum Free Media eliminate the need for this washing step

Savings


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Thermo Fisher Serum Free Media

VP-SFM

Sf-900 II

CD BHK-21

OptiPRO SFM

Designed for vaccine production in

Vero cells

Adherent/Microcarrier

Designed for vaccine production

in BHK-21 cells

Suspension

Designed for protein and vaccine

production in insect cells

Suspension

Broad range serum free medium suitable for

most kidney cells

Adherent/Microcarrier



Production Scale


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Bulk Harvest Virus Inoculation

+

Increase/Replace Medium

200L Bioreactor

Expansion

Typical upstream vaccine manufacturing processes

Cell Expansion Virus Production

Cell Thaw

Roller Bottle

Plant

Cell

Growth

Virus

Growth

Virus Inoculation

+

Replace Medium

Virus Seed

Bulk Harvest T-flasks

Transfer

Cell Factories

Cell

Growth

Virus

Growth

Cell Factory

Plant

Virus Seed

Virus Inoculation

+

Replace Medium

Bulk Harvest

Transfer

Spinner Flasks

Cell

Growth

Virus

Growth

50L Bioreactor

Plant

Virus Seed

Transfer Transfer


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Choice of vessel impacts capital as well as operational costs

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$0,5

$1,0

$1,5

$2,0

$2,5

0,01 1 100 10000

Co

ntr

ol U

nit

Exp

en

dit

ure

(U

SD)

Mill

ion

s

Production Scale (m2)

Roller Bottle Cell Factories Microcarrier

$0

$5.000

$10.000

$15.000

$20.000

$25.000

$30.000

Roller Bottle Cell Factories Microcarrier

Op

era

tin

g C

ost

s ($

/m2)

Scale = 10,000 m2

High capital costs make roller bottles impractical at

large scales

The cost estimates do not account for productivity

enhancements in bioreactors

Roller Bottles require more manipulation steps in

processes. Higher risk!

Cell Factories and Bioreactors offer superior floor

space consumption in the manufacturing suites

Simón, Marcos. 2015 Bioprocess International 13(8)


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Re-feed Optimization in Bioreactors Enhances Titer

Batch

No medium exchange

Bolus feeds of Glucose and

Glutamine during production

phase when depleted

Semi-Batch

After 2 days, replaced 33% of

medium daily

Bolus feeds of Glucose and

Glutamine on day 4

Perfusion

Half working volume

After 2 days full volume

replacement per day

Recirculation

5x reactor volume as total

medium volume

After day 1, recirculation at 5

volumes/day increased to 10

volumes/day on day 10

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Polio 1 Polio 2 Polio 3

D A

nti

gen

(D

U/m

L)

Batch Semi-Batch Perfusion Re-circulation

Thomassen et al. 2013 Vaccine 32(24)

Process development enables superior output without

other investments in technology


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CD BHK-21: Increased Productivity Through High Cell Density Process Development

Higher cell density yields greater antigen productivity

FM

D V

iral

Tit

er


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Thermo Fisher Nunc Cell Factories


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Thermo Fisher Nunc Cell Factories


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Thermo Fisher HyPerforma Single Use Bioreactors

50L 100L 250L 1000L 2000L


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Summary

Modern technologies allow substantial opportunities to enhance process output

Utilization of these technologies requires deliberate decisions on cell-line, medium and

production platform

Thermo Fisher Scientific products provide customers the ability to leverage process

improvements that may lower their product costs


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Building Lower Cost Upstream Vaccine Processes · Designed for vaccine production in Vero cells Adherent/Microcarrier Designed for vaccine production in BHK-21 cells Suspension Designed