Case Study: Formulation and St bilit Ch ll f Vi Stability Challenges for Virus Therapeutics: Above and Beyond Proteins Beyond Proteins Byeong S. Chang, PhD, Symyx Solutions Inc. Th V i D l tF S t b 23 2009 The V accine Development Forum, September 23, 2009
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Formulation and Stability Challenges for Virus Therapeutics
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
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Case Study: Formulation and St bilit Ch ll f ViStability Challenges for Virus
Therapeutics: Above and Beyond ProteinsBeyond Proteins
Byeong S. Chang, PhD, Symyx Solutions Inc.Th V i D l t F S t b 23 2009The Vaccine Development Forum, September 23, 2009
Outline
Introduction
Analytical Methods and Challenges
Formulation Development of Virus Products
Summaryy
Virus vs. Proteins
Virus vs. Proteins
Proteins Virusote s us
Size 3-10 nm 20-300 nm
C iti polypeptide/ Capsid proteins,Composition polypeptide/glycosylation
Capsid proteins, glycosylation, DNA
Structure Folded single molecule subunits
DNA coated with capsid proteinsmolecule - subunits capsid proteins
Conformation change Unfolding
Transition for infection, multiplication, di i ti *change dissociation *
Activity Molecular level Cellular level
* Adapt to environmental change
Analytical Methods and Challenges
Proteins> HPLC analyses (SEC IEX RP HIC etc)> HPLC analyses (SEC, IEX, RP, HIC, etc)> 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> Vector titer in particles/mL> Infectivity> Genomic structure, PCR> Antigenicity> Antigenicity
Product concentration> Low concentration> Low concentration> Different concentrations of component proteins
Heterogeneity of solutionHeterogeneity of solution> Active aggregation> Preferential surface adsorption> Cause for experimental errors> Cause for experimental errors
Stability during sample preparation & characterizationcharacterization
Correlation between biochemical results and biological activitiesbiological activities
Uneven distribution in solution
Effect of Diluent on Stability
4.0E+13
2.0E+13
3.0E+13
rticl
es/m
L
1.0E+13
Par
0.0E+00
LB PBS Water
Effect of diluent on Stability
4x1015
5x1015 Dilution with PBS Dilution with LB
2x1015
3x1015
Tite
r/mL
1x1015
2x10
0 2 4 6 8
0
Storage (weeks)
Significant titer is lost almost instantly if wrong diluent is used. Titer is also decreasing rapidly during analysis.
Inconsistent Recovery by HPLC
16
20
16
20
8
12
gnal
Inte
nsity
8
12
gnal
Inte
nsity
4
Sig
4
Sig
00 10 20 30 40 50 60 70
Time (min)
00 10 20 30 40 50 60 70
Time (min)
Inconsistent Recovery by HPLC
New diluent H2O
Diluent only water only
Retention Time (minutes)
Inconsistent Recovery by HPLC
Diluent A Diluent BDiluent A Diluent B
Retention Time (minutes)
Identifying proper diluent is critical for successful HPLC analyses.
Lack of Correlation anong Analytical Methods
RP-HPLC IEX-HPLC
Sign
al In
tens
ity
Control
Lyophilized
Control
y p
Liquid
Lyophilized
Liquid
0 10 20 30 40
Time (min)0 5 10 15
Time (min)
The integrity of major capsid proteins may be observed by the HPLC methods.g y j p p y y
Lack of Correlation anong Analytical Methods
SDS-PAGE
trol
philiz
ed 1
philiz
ed 2
id 1
id 2
All forulations appear to have similar quantity of capsid proteins
Con
Lyop
Lyop
Liqu
Liqu
All forulations appear to have similar quantity of capsid proteins.
Lack of Correlation anong Analytical Methods
Form
Recovery after storage (%)
Particles Infectious Units Potencyy
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 bioassaysonly observed by bioassays.
Formulation Development
Major stress factors
Observed degradation pathways
Stabilit Indicating Assa sStability Indicating Assays
Effect of basic formulation parameters
Major Stress Factors
Freeze-thawing
Frozen Temp (°C) -70 -20Temp ( C)
Activity (%) 0 100
Effect of Formulation on Stability
pH (with ionic tonicity modifier)
100
60
80
100
pH7y(%)
20
40
60 pH 7
pH 6
pH 5Recovery
0
20
SEC IEX RP
Effect of Formulation on Stability
Ionic Strength (at pH 7)
100
60
80
Ionicry (%
)
20
40
Ionic
Non‐ionic
Rec
over
0
4°C 25°C
Effect of Formulation on Stability
Liquid vs. Lyophilized ( ft 6 th t t t )(after 6 months at room temperature)
100
90
95
100
y(%
)
85
90Lyophilized
LiquidRecovery
75
80
SEC RP IEX
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 fformulation 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.