Case Study from Vaccine Development Forum in 2009. Discussion of challenges of virus formulations vs. protein formulations. Includes a comparison of analytical methods and formulation solutions

1. Case Study: Formulation and
Stability Ch ll
St bilit Challenges for Virus
f Vi
Therapeutics: Above and
Beyond Proteins
Byeong S. Chang, PhD, Symyx Solutions Inc.
The Vaccine Development F
Th V i D l t Forum, S t b 23 2009
September 23,

2. Outline
Introduction
Analytical Methods and Challenges
Formulation Development of Virus Products
y
Summary

3. Virus vs. Proteins

4. Virus vs. Proteins
Proteins
ote s Virus
us
Size 3-10 nm 20-300 nm
polypeptide/ Capsid proteins,
Composition
C iti glycosylation glycosylation, DNA
Folded single DNA coated with
Structure molecule - subunits capsid proteins
Transition for infection,
Conformation
Unfolding multiplication,
change dissociation *
di i ti
Activity Molecular level Cellular level
* Adapt to environmental change

5. Analytical Methods and Challenges
Proteins
> HPLC analyses (SEC IEX RP HIC etc)
(SEC, IEX, RP, HIC,
> Electrophoresis (CE, SDS-PAGE, IEF)
> Peptide mapping, AAA, Mass spectrometry
> Str ct ral anal ses (CD, Fl orescence FTIR
Structural analyses (CD Fluorescence, FTIR,
DSC etc.)
> Bioassays
Viral particles
> Plaque-forming unit (pfu) assay
> Vector titer in particles/mL
> Infectivity
> Genomic structure, PCR
> Antigenicity

6. Searching for Biochemical markers for Virus
HPLC analyses
> SEC-HPLC
> IEX-HPLC
> RP-HPLC
Electrophoresis
> SDS-PAGE (Western Blot)
SDS PAGE

7. SEC-HPLC
Signal Intensity
y
After Stress
Control
0 5 10 15 20 25 30
Time (min)

8. IEX-HPLC
sity
Signal Intens
Stressed
S
Control
0
0 10 20 30
Time (min)

9. IEX-HPLC
Signal Intensity
Retention TIme (Min)

10. RP-HPLC
Stressed
ty
gnal Intensit
Sig
Control
0 10 20 30 40 50 60
Time (min)

11. SDS-PAGE with Western Blot
116.3
97.4
66.3
55.4

12. Analytical Challenges
Product concentration
> Low concentration
> Different concentrations of component proteins
Heterogeneity of solution
> Active aggregation
> Preferential surface adsorption
> Cause for experimental errors
Stability during sample preparation &
characterization
Correlation between biochemical results and
biological activities

13. Uneven distribution in solution

14. Effect of Diluent on Stability
4.0E+13
3.0E+13
rticles/mL
2.0E+13
Par
1.0E+13
0.0E+00
LB PBS Water

15. Effect of diluent on Stability
15
5x10 Dilution with PBS
Dilution with LB
15
4x10
Titer/mL
15
3x10
15
2x10
15
1x10
0
0 2 4 6 8
Storage (weeks)
Significant titer is lost almost instantly if wrong diluent is used. Titer is also
decreasing rapidly during analysis.

16. Inconsistent Recovery by HPLC
20 20
16 16
gnal Intensity
gnal Intensity
12 12
8 8
Sig
Sig
4 4
0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Time (min) Time (min)

17. Inconsistent Recovery by HPLC
New diluent H2O
Diluent only water only
Retention Time (minutes)

18. Inconsistent Recovery by HPLC
Diluent A Diluent B
Retention Time (minutes)
Identifying proper diluent is critical for successful HPLC analyses.

19. Lack of Correlation anong Analytical Methods
RP-HPLC IEX-HPLC
Signal Intensity
Control
Control
Lyophilized
y p
Lyophilized
Liquid
Liquid
0 10 20 30 40 0 5 10 15
Time (min)
Time (min)
The integrity of major capsid p
g y j p proteins may be observed by the HPLC methods.
y y

20. Lack of Correlation anong Analytical Methods
SDS-PAGE
philized 1
philized 2
Liquid 1
Liquid 2
Control
Lyop
Lyop
All forulations appear to have similar quantity of capsid proteins
proteins.

21. Lack of Correlation anong Analytical Methods
Recovery after storage (%)
Form Particles Infectious Units Potency
y
4°C 25°C 4°C 25°C 4°C 25°C
Liquid 100 95 99 91 129 4
Lyophilized 76 86 37 29 49 18
Integrity of minor, but critical for the biological fucntion, proteins may be
only observed by bioassays.
bioassays

22. Formulation Development
Major stress factors
Observed degradation pathways
Stability
Stabilit Indicating Assa s
Assays
Effect of basic formulation parameters

23. Major Stress Factors
Freeze-thawing
Frozen
-70 -20
Temp (°C)
( C)
Activity (%) 0 100

24. Effect of Formulation on Stability
pH (with ionic tonicity modifier)
100
80
Recovery (%)
y
60 pH 7
pH 7
pH 6
40
pH 5
20
0
SEC IEX RP

25. Effect of Formulation on Stability
Ionic Strength (at pH 7)
100
80
Recover (%)
60
ry
Ionic
Non‐ionic
40
20
0
4°C 25°C

26. Effect of Formulation on Stability
Liquid vs. Lyophilized
(after
( ft 6 months at room temperature)
th t t t )
100
95
Recovery (%)
y
90
Lyophilized
85 Liquid
80
75
SEC RP IEX

27. Summary
Orthogonal biochemical analyses can be developed for
monitoring the integrity of capsid.
g g y p
Spontaneous adaptation of viral particles to environment
requires careful qualification of analytical process.
General formulation parameters critical for protein
therapeutics appear to be critical for viruses, too.
Contact surfaces need to be confirmed for analytical and
formulation purposes.
Stability profile of viral particles can be different from
proteins (stresses, key degradation pathways).
Biochemical markers may not always represent specific
viability of virus therapeutics.