Analytical Methods for Use in the Characterization and

Release of Gene Therapy Products

American Society of Gene & Cell TherapyTranslational Science Training Course: Bench to Bedside –

Facilitating First in Human TrialsMay 17, 2011

Phillip Ramsey

Analytical Method needs for Gene Therapy Products

• Characterization • Process Monitoring • Release Testing• Stability Assessment

• Discovery• Non-clinical• Pre-clinical• Exploratory IND • Phase I• Phase II• Phase III• Phase IV/Post

Marketing

Requirements for Assessing Product

• Identity • Purity• Potency• Safety• Strength/dose• (Performance)

Ad Vaccine Technology Platform

Ad Based Expression Vectors

• Ad5 Vector System- GenVec 293ORF6(HIV)- Crucell PER.C6 (Ebola)

• Other- Alternative serotype- Modified Vectors

Immunogens• HIV

- Clade A,B,C/modified env- gag, pol, nef

• Ebola- NP, GP (strain-specific)

• SARS- Spike protein

• Influenza - HA, M2, NP

Viral Vector

CandidateAd Vector

ImmunogenGene(s)

Vaccine Research Center, NIAID

DNA Vaccine Technology Platform

DNA Expression Vectors

• Enhancer/Promoter• Poly (A)

– Terminate expression• Antibiotic Resistance

– Selectable marker• Plasmid Backbone

Immunogens• HIV

– Clade A,B,C/modified env– gag, pol, nef

• Ebola– NP, GP (strain-specific)

• SARS– Spike protein

• West Nile Virus– Envelope protein

• Influenza– HA, M2, NP

ExpressionVector

CandidatePlasmidVaccine Immunogen

Gene(s)

Vaccine Research Center, NIAID

Characterization

LinearLinear

Open-circularOpen-circular

SupercoiledSupercoiled

Process Monitoring Seph 4FF Adeno Eng 11Mar09:10_UV1:131_280nm Seph 4FF Adeno Eng 11Mar09:10_UV2:131_254nm Seph 4FF Adeno Eng 11Mar09:10_UV3:131_215nm Seph 4FF Adeno Eng 11Mar09:10_Cond_102 Seph 4FF Adeno Eng 11Mar09:10_Fractions

0.0

0.2

0.4

0.6

0.8

1.0

1.2

AU

0.0

0.2

0.4

0.6

0.8

1.0

1.2

AU

12.0 13.0 14.0 15.0 16.0 lOutlet2 Outlet3 Outlet1

Release Testing

Identity

~2500 bp = VRC5736 and VRC57383030 bp = VRC57383761 bp = VRC57365039 bp = VRC44045886 bp = VRC44016338 bp = VRC57377344 bp = VRC4409

5039 bp = VRC4404

Age I + Cla I Band Sizes

Xho I Band Sizes

~3350 bp =

5886 bp = VRC44017344 bp = VRC4409

~2950 bp =

VRC5736VRC5737VRC5738

VRC5736VRC5737VRC5738

Purity

Minutes

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

RFU

0

2

4

6

8

10

12

RFU

0

2

4

6

8

10

12LIF - Channel 1TM_10ugmL_half dye

LIF - Channel 1VLP_TT9_ext_0.5psi_10s

Potency

Safety

Strength/Dose

Monitor for plaque formation

Performance

Stability Testing

Subset of your release testing.

75

80

85

90

95

100

% P

urity

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (Months)

Linear Fit

Example of ICH Q1E Evalutation of Stability Data

The intersection of thespecification and the 95%confidence interval is thepredicted shelf life.

Accelerated/Stressed Stability

Study DescriptionLong term ≤ -60°CAccelerated ≤ -20°CAccelerated 2°C to 8°CStress Condition

25°C ± 2°C

Stress Condition

38°C to 42°C

Freeze/Thaw Freeze material at ≤ -60°C overnight then store daily at 2°C to 8°C until material is thawed. Repeat this cycle for a total of 5 freeze / thaw cycles.

Protein Concentration of CHIKV VLP

01020304050607080

0 1 2 3 4 5

Weeks on Stability

Con

cent

ratio

n (u

g/m

L)

2-8°C23-27°C38-42°C

Characterization/Qualification/Validation

Analytical Method

Characteristics (Data Elements)

IdentificationLimit Testing for Impurities Assay

(Dissolution, measurement only; content/potency)Quantitative Qualitative

Accuracy No Yes No YesPrecision –

Repeatability No Yes No YesPrecision -

Intermediate No Yes(1) No Yes(1)

Specificity/ Selectivity(2) Yes Yes Yes Yes(4)

Detection Limit No No(3) Yes No

Quantitation Limit No Yes No No

Linearity No Yes No Yes

Range No Yes No Yes

Goal and Intended Purpose

Monitor product and product testing.

References

• FDA: http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/default.htm

• EMA -http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/landing/human_medicines_regulatory.jsp&murl=menus/regulations/regulations.jsp&mid=WC0b01ac058001ff89

• ICH - http://www.ich.org/home.html• USP - http://www.usp.org/