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

Searching for Biochemical markers for Virus

HPLC analyses> SEC-HPLC> IEX-HPLC> RP-HPLC

Electrophoresis> SDS-PAGE (Western Blot)SDS PAGE (Western Blot)

SEC-HPLC

yig

nal I

nten

sity

After Stress

S

Control

0 5 10 15 20 25 30

Time (min)

IEX-HPLC

sity

Sign

al In

tens

Stressed

S

Control

00 10 20 30

Time (min)

IEX-HPLC

Sig

nal I

nten

sity

Retention TIme (Min)

RP-HPLC

tyStressed

gnal

Inte

nsit

Sig

Control

0 10 20 30 40 50 60

Time (min)

SDS-PAGE with Western Blot

116.397.4

66.3

55.4

Analytical Challenges

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.