Analytical Methods for Use in the Characterization and ... · Analytical Methods for Use in the...
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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
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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
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Requirements for Assessing Product
• Identity • Purity• Potency• Safety• Strength/dose• (Performance)
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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
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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
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Characterization
LinearLinear
Open-circularOpen-circular
SupercoiledSupercoiled
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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
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Release Testing
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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
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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
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Potency
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Safety
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Strength/Dose
Monitor for plaque formation
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Performance
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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.
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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
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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
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Goal and Intended Purpose
Monitor product and product testing.
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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/