Interferon -1 Production in CHO Cells and Growth Optimization in Orbitally Shaken Bioreactors

FE 536 Design of Experiments Project Presentation by mran ZER, Damla TAYKOZ, Melis OLUM

Project Outline

Background Information Methods of the Study Results and Discussion Concluding Remarks

Multiple Sclerosis (MS) is an autoimmune disorder

Multiple sclerosis, progresses with demyelination of nerve tissue in the brain.

May be caused by genetic or environmental factors.

MS may affect several body functions

Interferon -1, is a frequently used drug molecule for MS disease FDA approved Annovex. drugs: Rebif, Cinnavex,

These drugs aim to relapse disease progression by reducing inflammation.

This protein may be produced as a recombinant product in various organisms such as bacteria or mice.

Production parameters matter

Protein production is a result of cell growth. In recombinant protein production, several factors directly affect the yield. Chemical inducer Growth factors Agitation Inhibitor concentration

Methods of Study
Chinese Hamster Ovary cells - in orbitally shaken bioreactors (50 ml). ProCHO5 medium - contains Fetal Bovine Serum (FBS). 5.105 cells/ml initial cell concentration. 36 hours of cultivation. Viable cells are counted with hamocytometer, to measure cell growth level.

Methods of Study
Factors Agitation speed (rpm) Serum Concentration (% of total working volume) Level 1 0 1 Level 2 40 5.5 Level 3 80 10

Inducer Concentration (ng/ml)

10

505

1000

Face centered central composite design. 31 runs analyzed with Design Expert 8.0 program. Regression model of the system was validated with extra runs.

Results and Discussion

Face-centered CCD contains 16 factorial terms, 12 axial terms and 3 center points.

Design Suggestion by Design Expert Program

Quadratic effect was suggested due to lack of fit test

ANOVA of Quadratic Design

The lack of fit is significant. This is an undesired situation for the analysis, therefore modifying the model by removing insignificant parameters is applied, but an insignificant lack of fit value for the test model could not be obtained. Therefore, second suggested model for the system that is cubic model using aliased terms was used.

ANOVA of Cubic Design and Main Effects by MINITAB 15

Main Effects Plot for Viable Cell Count
Data Means
A 2,0 1,5 1,0 B

Mean

0,5 -1 2,0 1,5 1,0 0,5 -1 0 1 0 C 1 -1 0 1

Interactions Plots
Design-Expert Software Factor Coding: Coded Viable cell count CI Bands Design Points
V ia b le c e ll c o u n t
4

Interaction
B: agitation

Design-Expert Software Factor Coding: Coded Viable cell count CI Bands Design Points
4

Interaction
B: agitation

Design-Expert Software Factor Coding: Actual Viable cell count CI Bands Design Points
4

Interaction
C: inducer

Design-Expert Software Factor Coding: Actual Viable cell count CI Bands Design Points X1 = B: agitation X2 = C: inducer Actual Factor A: serum = 0.00
4

Interaction
C: inducer

V ia b le c e ll c o u n t

X1 = A: serum X2 = B: agitation Coded Factor C: inducer = 0.000

V ia b le c e ll c o u n t

X1 = A: serum X2 = B: agitation Coded Factor C: inducer = -1.000 B- -1.00 B+ 1.00

X1 = B: agitation X2 = C: inducer

V ia b le c e ll c o u n t

2 3 2
2

Actual Factor A: serum = -1.68 C- -1.68 C+ 1.68

2 3 2
2

B- -1.00 B+ 1.00

C- -1.68 C+ 1.68

2 2

2
0
0
0

2 2

-1

-1

-1
-1.68 -0.84 0.00 0.84 1.68

-1

-1.68

-0.84

0.00

0.84

1.68

-1.00

-0.50

0.00

0.50

1.00

-1.00

-0.50

0.00

0.50

1.00

A: serum
Design-Expert Software Factor Coding: Coded Viable cell count CI Bands Design Points X1 = A: serum X2 = B: agitation Coded Factor C: inducer = 1.000 B- -1.00 B+ 1.00
V ia b le c e ll c o u n t
4

A: serum

B: agitation

B: agitation

Interaction
B: agitation

Design-Expert Software Factor Coding: Actual Viable cell count CI Bands Design Points
4

Interaction
C: inducer

X1 = B: agitation X2 = C: inducer Actual Factor A: serum = 1.68

V ia b le c e ll c o u n t

C- -1.68 C+ 1.68

2
0

-1

-1
-1.00 -0.50 0.00 0.50 1.00

-1.68

-0.84

0.00

0.84

1.68

A: serum

B: agitation

AB interaction is more significant than BC interaction because AB lines intersect each other while BC lines have less intersection. ANOVA p values also confirm the evaluation about the interaction.

Regression Analysis
Regression formula that is based on significant term generated by the Design Expert software is given in Equation below: = 2,44 + 0,43 + 0,73 0,30 + 0,10 0,052 + 0,066 2 1,13 2 1,02 2 0,54 2 + 0,15 2 (0,36 2 )

Residual Analysis
Design-Expert Software Viable cell count Color points by value of Viable cell count: 2.95 0.0125

Normal Plot of Residuals

N o r m a l % P r o b a b ility
99 95 90 80 70 50 30 20 10 5 1

-2.00

-1.00

0.00

1.00

2.00

Internally Studentized Residuals

Residuals are normally distributed because all the points are very close to the centerline except the first and the last runs.

Design-Expert Software Viable cell count Color points by value of Viable cell count: 2.95 0.0125

In te r n a lly S tu d e n tiz e d R e s id u a ls

Residuals vs. Run

3.00

2.00

1.00

0.00

-1.00

-2.00

-3.00

11

16

21

26

31

There is no particular pattern in the residual distribution.

Run Number

Design-Expert Software Viable cell count Color points by value of Viable cell count: 2.95 0.0125
3.00

Predicted vs. Actual

2.50

P r e d ic te d

2.00

1.50

2
1.00

0.50

0.00

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Actual

Model adequately represents the system.

Optimization
Design-Expert Software Factor Coding: Coded Viable cell count Design Points 2.95 0.0125 X1 = A: serum X2 = B: agitation Coded Factor C: inducer = -1.000
0.50
a g ita tio n

1.00 2

Viable cell count

1

Design-Expert Software Factor Coding: Coded Viable cell count Design points above predicted value Design points below predicted value 2.95 0.0125 X1 = A: serum X2 = B: agitation
c o u n t c e ll

1.5 2
0.00

Coded Factor C: inducer = -1.000

4 3 2 1 0 -1 1.00

V ia b le

B :

-0.50

1 0.5

1.00 0.50 0.00

0.50

0.00

0
-1.00 2 -1.00 -0.50 0.00 0.50

2
1.00

-0.50

-0.50

A: serum

B: agitation

-1.00 -1.00

A: serum

@ low level of inducer.

Design-Expert Software Factor Coding: Coded Viable cell count Design Points 2.95 0.0125 X1 = A: serum X2 = B: agitation Coded Factor C: inducer = 0.000

1.00

Viable 2cell count

2

Design-Expert Software Factor Coding: Coded 2 Viable cell count Design points above predicted value Design points below predicted value 2.95 0.0125

0.50
a g ita tio n

X1 = A: serum X2 = B: agitation

2.5
0.00 2

Coded Factor C: inducer = 0.000

c o u n t c e ll

4 3 2 1 0 -1 1.00

B :

2
-0.50

1.5 1
-1.00 -1.00 -0.50

V ia b le

1.00 0.50
0.50 1.00

0.50

2
0.00

0.00 0.00 -0.50 -0.50

A: serum

A: serum

B: agitation

-1.00 -1.00

@ medium level of inducer.

Design-Expert Software Factor Coding: Coded Viable cell count Design Points 2.95 0.0125 X1 = A: serum X2 = B: agitation Coded Factor C: inducer = 1.000

1.00 2

Viable cell count

0.5

Design-Expert Software Factor Coding: Coded Viable cell count Design points above predicted value Design points below predicted value 2.95 0.0125

0.50
a g ita tio n

X1 = A: serum X2 = B: agitation Coded Factor C: inducer = 1.000

c o u n t c e ll

1
0.00

4 3 2 1 0 -1 1.00

1.5
2

B :

-0.50

0.5 0

V ia b le

1.00 0.50

0.50

-0.5
-1.00 2 -1.00 -0.50 0.00 0.50

0.00 0.00 -0.50 -0.50

2
1.00

A: serum

B: agitation
A: serum

-1.00 -1.00

@ high level of inducer.

Numerical Optimization

Validation
Coded Terms Runs A B C Serum Actual Terms Agitation Inducer Runs 1 2 3 4 5 Average Predicted value 1,206 0,906 1,516 2,066 1,076 1,354 Observed value 1,25 0,975 1,5 2 1,125 1,37 Error 4% 8% 1% 3% 5% 2%

1
2 3 4 5

-1
-1 -1 +1 +1

0
0 +1 -1 0

-1
+1 0 0 -1

1
1 1 10 10

40
40 80 40 40

10
1000 505 10 10

The error is 2% throughout the experiment and it is thought that this error
value is an acceptable low value with respect to the literature optimization values (Tissot 2011).

Significant Effects
This study should have done by measuring two responses that are viable cell count and recombinant protein concentration. Because of inadequate number of ELISA kits, recombinant protein concentration measuring could not be completed. Therefore, system is analyzed by using only one response that is viable cell count. The results had a normal distribution and each main factor was shown to have significantly different effects on cell growth. Agitation speed and inducer concentration had two-sided effects on the yield, so an optimization for these two factors had great importance for a maximized cell growth. On the other hand, serum concentration factor had a comparably linear effect on the yield. Serum concentration-agitation speed interaction (AC) and agitation speed-inducer concentration interactions (BC) have significant effects on the yield.

Optimized Parameters
This study shows that recombinant Chinese Hamster Ovary (CHO) cells were
cultured best at 10% serum concentration, 40 rpm agitation speed and 505 ng/ml inducer concentration at small scale bioprocess to achieve optimum viable cell count in production of recombinant interferon - 1. Therefore more than one best level may have been obtained for particularly one desired target.

Validation and Precision

As a result of regression analysis and validation runs, study was completed with %2 error. This is a very small error level for biological systems because, there are usually many more factors that cannot be controlled such as pressure or humidity changes in the environment, human-related mistakes, aging or origin of cells.

Therefore, it can be concluded that our model fits well for this experimental set-up and have high precision.

Novelty and Importance

This study reveals optimized results for interferon 1 production in recombinant CHO cells and is the first optimization study for recombinant interferon -1 production in CHO cells by using single use bioreactors at small- scale.

This study also promises new, small-scale animal cell culture production technology in single use bioreactors that is concluded as an efficient method.

Additionally, these optimization results belong to small-scale production of recombinant interferon -1. These results cannot be generalized to different scale production due to exploitation of other process parameters but they may lead another optimization studies in large-scale production.

References
Jacobs LD, Cookfair DL, Rudick RA, Herndon RM, Richert JR, Salazar AM, et al. Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG). Ann Neurol. 1996;39(3):285-94. Tenembaum SN, Banwell B, Pohl D, Krupp LB, Boyko A, Meinel M, et al. Subcutaneous Interferon Beta-1a in Pediatric Multiple Sclerosis: A Retrospective Study. J Child Neurol. 2013;10:10. Shekhter, II, Beiko VP, Bulenkov MT, Khodova OM, Kolevatykh MA, Izotova LS, et al. [Obtaining human recombinant (serine-17) beta-interferon by the method of oligonucleotide-directed mutagenesis and its expression in Escherichia coli]. Antibiot Khimioter. 1991;36(8):25-8. Abdul-Ahad AK, Galazka AR, Revel M, Biffoni M, Borden EC. Incidence of antibodies to interferon-beta in patients treated with recombinant human interferon-beta 1a from mammalian cells. Cytokines Cell Mol Ther. 1997;3(1):27-32. Houdebine LM. Production of pharmaceutical proteins by transgenic animals. Comp Immunol Microbiol Infect Dis. 2009;32(2):107-21. Kieseier BC, Calabresi PA. PEGylation of interferon-beta-1a: a promising strategy in multiple sclerosis. CNS Drugs. 2012;26(3):205-14. Zhang X, Stettler M, De Sanctis D, Perrone M, Parolini N, Discacciati M, De Jesus M, Hacker D, Quarteroni A, Wurm F, Use of orbital shaken disposable bioreactors for Mammalian cell cultures from the milliliter-scale to the 1,000-liter scale. Adv Biochem Eng Biotechnol. 2010;115: 33-53. Ikura K, Nagao M, Masuda S, Sasaki R, Animal Cell Technology: Challenges for the 21st Century. Kluwer Academic Publishers, 1998; 314-387. Tissot S, OrbShake Bioreactors for Mammalian Cell Cultures: Engineering and Scale-up,2011, EPFL (Ecole Polytechnique Federale de Lausanne) PhD Thesis.