DOE case: Virus concentration

Hans de Hoog

DOE case: Virus concentration

Intervet-SP Animal Health, Boxmeer

Presentation outline

•

Introduction Intervet SPAH

•

Case study, part 1



PEG precipitation

•

Case study, part 2



buffer optimisation

•

DOE



design and analysis

•

Conclusions

Intervet Schering-Plough Animal Health

We develop, manufacture and market a broad range of veterinary medicines

and services for the prevention, treatment and control of disease in all major

farm and companion animal species.

Products:

• Vaccines

• Pharmaceuticals (anti-infectives, anti-paracitic, fertility treatment)

• Innovative animal health programs

Viral-Bioprocess Technology & Support (BTS-V)

Research

Production

Trouble shooting

‘Best of both worlds’

BTS-V

Production systems

Bioreactor

(2-2000L)

Roller bottles

(max. 2000/batch)

Cell factories

Bioreactors

2-2000L

Case study: objective

•

Virus concentration (and purification)

•

Current yield 100 AU/ml



request 400



target 1000

AU/ml

Part 1, PEG precipitation

•

Use of Polyethylene Glycol (PEG) for precipitation

Results

•

Factors tested: [PEG], chain length, [salt], chloroform, pH,

solubilisation medium

•

Method: screening designs (FFD, data not shown)

•

Important factors found: [PEG], [salt] and pH

•

Recovery antigenic mass >80%

•

Protein loss (purification)

±

85%

Part 2: Solubilisation buffer

•

DOE



optimisation of factors Phosphate, NaCl, Surfactant and pH

•

Model choice



Central Composite Design, 28 runs

•

Measurement of yield (AM recovery) and protein removal (%)

•

Factor levels

Factor

Level

Log2

Phosphate (Molar)

0.2

-2.32

0.1

-3.32

0.05

-4.32

level

Log5

Surfactant (%)

0.25

-0.86

0.05

-1.86

0.01

-2.86

level

linear

NaCl (Molar)

0.1

-0.25

Importance of randomisation

Design-Expert® Software

Antig. Mass

Color points by value of

Antig. Mass:

1264.5

111.3

Run Number

Int

er

nal

ly

S

tudent

iz

ed

R

es

idual

s

Residuals vs. Run

-3.00 -1.50 0.00 1.50 3.00

1 5 9 13 17 21 25 29 33

Run

1st

2nd

Difference

1

1094

869

21%

2

1145

859

25%

3

1139

943

17%

4

1265

872

31%

5

671

510

24%

6

1058

812

23%

7

1146

993

13%

8

1239

964

22%

9

1038

731

30%

10

1125

825

27%

11

1166

800

31%

Design-Expert® Software

AM

Color points by value of

AM:

1037.8

111.3

Run Number

Int

er

nal

ly

S

tudent

iz

ed

R

es

idual

s

Residuals vs. Run

-3.00 -1.50 0.00 1.50 3.00

1 5 9 13 17 21 25 29 33

ANOVA, AM recovery

Sum of Mean F p-value Source Squares df Square Value Prob > F

Model 14113 10 1411.2997 7.729798 < 0.0001 A-Phosphate 5436.178 1 5436.1780 29.77437 < 0.0001 B-NaCl 1867.749 1 1867.7489 10.22981 0.0043

C-Surfactant 210.8175 1 210.8175 1.154664 0.2948

D-pH 4654.272 1 4654.2718 25.49181 < 0.0001

AB 409.5364 1 409.5364 2.243063 0.1491 AC 682.6782 1 682.6782 3.739081 0.0667

AD 1300.193 1 1300.1930 7.121258 0.0144

BC 24.71399 1 24.7140 0.13536 0.7166

BD 994.5617 1 994.5617 5.447292 0.0296

CD 2.121853 1 2.1219 0.011622 0.9152 Residual 3834.161 21 182.5791

Lack of Fit 1593.684 13 122.5911 0.437732 0.9106 Pure Error 2240.478 8 280.0597

Cor Total 17947.16 31

Std. Dev. 13.51218 R-Squared 0.7864 Mean 70.24374 Adj R-Squared 0.6846 C.V. % 19.23614 Pred R-Squared 0.4997 PRESS 8978.401 Adeq Precision 11.6006

Perturbation plot

Design-Expert® Software

AM recovery

AM recovery

Actual Factors

A: Phosphate = -3.34

B: NaCl = 0.25

C: Surfactant = -1.84

D: pH = 7.00

Perturbation

Deviation from Reference Point (Coded Units)

A

M

r

ec

ov

er

y

-1.000 -0.500 0.000 0.500 1.000 11.0 34.3 57.5 80.8 104.0

A

A

B

B

C

C

D

D

Response surface plot

Design-Expert® Software

AM recovery

Design Points

103.784

11.1327

X1 = A: Phosphate

X2 = D: pH

Actual Factors

B: NaCl = 0.25

C: Surfactant = -1.84

-4.32 -3.83 -3.34 -2.84 -2.35 6.50 6.75 7.00 7.25 7.50

AM recovery

A: Phosphate

D:

p

H

Response surface plot

Design-Expert® Software

AM recovery

Design Points

103.784

11.1327

X1 = A: Phosphate

X2 = C: Surfactant

Actual Factors

B: NaCl = 0.10

D: pH = 6.50

-4.32 -3.83 -3.34 -2.84 -2.35 -2.82 -2.33 -1.84 -1.35 -0.86

AM recovery

A: Phosphate

C

:

S

ur

fac

tant

ANOVA, Ratio AM/protein

Sum of Mean F p-value Source Squares df Square Value Prob > F

Model 1.090605 9 0.12117837 6.730271 0.0001 A-Phosphate 0.133851 1 0.133850722 7.434096 0.0123

B-NaCl 0.045457 1 0.045456573 2.524667 0.1263

C-Surfactant 0.162942 1 0.16294154 9.049806 0.0065 D-pH 0.421704 1 0.421704353 23.42154 < 0.0001

AB 0.049252 1 0.049251778 2.735454 0.1123 AC 0.038973 1 0.038973129 2.164575 0.1554 AD 0.06841 1 0.068409857 3.799497 0.0641

BD 0.132099 1 0.132098972 7.336804 0.0128 A^2 0.080628 1 0.080627566 4.478071 0.0459

Residual 0.396109 22 0.018004976

Lack of Fit 0.209482 14 0.014962965 0.641403 0.7767 Pure Error 0.186628 8 0.023328495

Cor Total 1.486715 31

Std. Dev. 0.134183 R-Squared 0.7336 Mean 0.680897 Adj R-Squared 0.6246 C.V. % 19.70674 Pred R-Squared 0.4391 PRESS 0.833854 Adeq Precision 11.5939

Response surface plot

Design-Expert® Software

Ratio AM/Protein

Design Points

1.01845

0.146601

X1 = A: Phosphate

X2 = D: pH

Actual Factors

B: NaCl = 0.10

C: Surfactant = -0.86

-4.32 -3.83 -3.34 -2.84 -2.35 6.50 6.75 7.00 7.25 7.50

Ratio AM/Protein

A: Phosphate

D:

p

H

Conclusions

•

Part 1, Precipitation



Important factors: [PEG], [Salt], [surfactant], pH

•

Problem



sedimentation

•

Part 2, Buffer optimization



>80% AM recovery, high purity

•

Clear solution



no sedimentation!

•

Target AM exceeded

Thank you

Questions?

Hans de Hoog

Titel: Virus concentration

Abstract:

Precipitation with polyethylene glycol (PEG) is a commonly used method for protein

removal from serum or other solutions. It can also be used to precipitate virusses in

cell culture harvests. In this case study, DOE was used as a screening tool to

optimize the precipitation process of a virus. After precipitation, the precipitate

needs to be dissolved for further use. An optimization design was used to obtain a

virus concentrate that was fully dissolved.