Gene Therapy and AAV: Advancing Analytical Characterization to Improve Product Understanding, Control and Comparability Exercises
CMC Strategy Forum17 July 2017
Herb Runnels, PhD
Pfizer, BioTx Pharmaceutical Sciences, Analytical R&D
Pfizer Proprietary
Practical Challenges:• Attribute criticality – field still in early understanding• Sample retain limitations due to small batch sizes• Limitations of standard assays – poor sensitivity, high
variability, technology availability
Comparability
Advancement of assays can improve attribute and product understanding, thus improving
comparability exercises
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• Introduction of New Assays: Mass Spec, RP-HPLC, Peptide Mapping and SEC-HPLC
• Case Study of Comparability Finding due to New Analytical Capabilities
• Importance and Challenge of Strong Potency Assay
• Residual Host Cell DNA Characterization: Qualitative PCR
• Improved Characterization (Full / Empty Ratio): EM and AUC
Presentation Outline
Pfizer Proprietary
Characterization and Comparability of AAV Capsid Proteins
• Intact Capsid Protein Analysis– Denature capsid and separate by RP chromatography prior to MS (RP-
HPLC/MS)
• Proteolytic Digest of Capsid Proteins (Peptide Mapping)– Denature capsid and digest with proteolytic enzyme prior to RP-HPLC-MS/MS
Capabilities of Mass Spec-Based Methods• Confirm amino acid sequence• Monitor clips/truncations• Determine cysteine oxidation state• Identify capsid protein modifications• Characterize unknowns/impurities
Mass Spectrometry
Pfizer Proprietary
AU
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Minutes36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40 41.60 41.80 42.00 42.20 42.40 42.60 42.80 43.00
AU
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
Minutes34.20 34.40 34.60 34.80 35.00 35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40
VP2
VP3
VP1VP1VP2VP3
Traditional: SDS-PAGE Purity Advanced: RP-HPLC Purity
AAV Serotype “A”
VP2 VP1
VP3AAV Serotype “B”
More quantitative Greater sensitivity
Less resource intensive Amenable to characterization by on-line mass spectrometry
Advancement of Traditional Purity Assay
Pfizer Proprietary
AU
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
0.34
0.36
0.38
Minutes10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00 170.00 180.00
Traditional: Western Blot Advanced: Peptide Mapping
Less resource intensive Platform assay for different serotypes
Amenable to characterization by mass spectrometry Increased ability to distinguish highly similar sequences
Vector Capsid Identity:
6
VP1
VP2
VP3
AAV Serotype “B”
Advancement of Traditional Assays
Sequence Coverage (98.6%)
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Advancement of Traditional Assays
Traditional: ELISA
Advanced: SEC-HPLC
Less variability Less resource intensive
Platform assay for different serotypes
Vector Particle Titer:
7
1.00E+13
5.10E+14
1.01E+15
1.51E+15
2.01E+15
2.51E+15
3.01E+15
0 2000000 4000000 6000000 8000000 10000000 12000000 14000000
Linear from 1.0E+13 – 2.5E+15 vp/mL
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
0.100
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
AAV MonomerParticle Aggregates
Pfizer Proprietary
Case Study
Denaturation
38.5
39
38.8
28
39.1
61
AU
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
0.080
Minutes35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40 41.60 41.80 42.00 42.20 42.40 42.60 42.80 43.00 43.20 43.40
VP2 VP1 VP360 capsid protein subunits (VP1,VP2,VP3) assembled into icosahedron at a ratio of 1:1:10
The RP-HPLC assay is a denaturing assay
RP-HPLC Method:
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Case Study
41.6
06
43.2
83
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes36.00 37.00 38.00 39.00 40.00 41.00 42.00 43.00 44.00 45.00 46.00 47.00 48.00
37.4
39
39.0
71
AU
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
Minutes31.00 31.50 32.00 32.50 33.00 33.50 34.00 34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00 40.50 41.00 41.50 42.00 42.50 43.00 43.50 44.00
39.1
61
AU
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
0.080
Minutes35.20 35.40 35.60 35.80 36.00 36.20 36.40 36.60 36.80 37.00 37.20 37.40 37.60 37.80 38.00 38.20 38.40 38.60 38.80 39.00 39.20 39.40 39.60 39.80 40.00 40.20 40.40 40.60 40.80 41.00 41.20 41.40 41.60 41.80 42.00 42.20 42.40 42.60 42.80 43.00 43.20 43.40
38.5
34
AU
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
0.070
0.075
0.080
Minutes34.50 35.00 35.50 36.00 36.50 37.00 37.50 38.00 38.50 39.00 39.50 40.00 40.50 41.00 41.50 42.00
Impurity Impurity
Process 1 - 1 Process 1 - 2
Process 2 - 1 Process 2 - 2
Identity of the impurity determined by MS with Heightened Characterization
New impurity was detected by RP-HPLC following process change
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Case Study
The new impurity observed by RP-HPLC was NOT detected in SDS-PAGE
MWM Process 2 Process 1
100 kDa75 kDa
50 kDa
37 kDa
VP1
VP3VP2
SDS-PAGE Method:
Pfizer Proprietary
Case Study
RP-HPLC coupled to MS determines the identity of the impurity Knowledge is Power in understanding how to control the process
Intact Protein
RP-HPLC/MS
Process 1
Process 2
RP-HPLC/MS Method:
Pfizer Proprietary
Case StudyA
U
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00
--- Batch 1--- Batch 3
The SEC-HPLC is a non-denaturing assay No difference in particle aggregates was observed between Process 1 and 2
Impurity is not inter-particle aggregates
Inter-particle multimers
Process 1Process 2
SEC-HPLC Method:
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Case Study
Small-scale DOE identifies causative step
AU
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
Minutes34.00 35.00 36.00 37.00 38.00 39.00 40.00 41.00 42.00 43.00 44.00 45.00 46.00 47.00 48.00 49.00 50.00 51.00 52.00
Process Condition 1
Process Condition 2
Process Condition 1b
RP-HPLC Method:
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Case Study
RT: 39.7 - 48.7
40 41 42 43 44 45 46 47 48Time (min)
0
20
40
60
80
100 NL: 6.10E6m/z= 232.2903-232.2927+278.5470-278.5498+347.9320-347.9354+463.5735-463.5781+694.8566-694.8636+1388.7059-1388.7197 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20170315TWP03NL: 6.10E6m/z= 232.4543-232.4567+278.7438-278.7466+348.1780-348.1814+463.9015-463.9061+695.3486-695.3556+1389.6900-1389.7038 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20170315TWP03
Process 2-2
RT: 39.7 - 48.7
40 41 42 43 44 45 46 47 48Time (min)
0
20
40
60
80
100 NL: 1.70E7m/z= 232.2903-232.2927+278.5470-278.5498+347.9320-347.9354+463.5735-463.5781+694.8566-694.8636+1388.7059-1388.7197 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP02NL: 1.70E7m/z= 232.4543-232.4567+278.7438-278.7466+348.1780-348.1814+463.9015-463.9061+695.3486-695.3556+1389.6900-1389.7038 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP02
Process 1-1
RT: 39.7 - 48.7
40 41 42 43 44 45 46 47 48Time (min)
0
20
40
60
80
100 NL: 1.40E7m/z= 232.2903-232.2927+278.5470-278.5498+347.9320-347.9354+463.5735-463.5781+694.8566-694.8636+1388.7059-1388.7197 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP04NL: 1.40E7m/z= 232.4543-232.4567+278.7438-278.7466+348.1780-348.1814+463.9015-463.9061+695.3486-695.3556+1389.6900-1389.7038 F: FTMS + p ESI Full ms [235.00-2000.00] MS 20160209TWP04
Process 2-1
23.0% Deamidation
42.6% Deamidation
46.4% Deamidation
Peptide map-MS also revealed higher deamidation levels in Process 2
Peptide Mapping
RP-HPLC-MS/MSBesides the impurity, an additional change was discovered:
RP-HPLC-MS/MS Method:
Deamidation:
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Case Study: Summary
Process was changed to include new step and unit operation Newly implemented purity method (RP-HPLC) showed a new impurity after
this change Traditional purity method (SDS-PAGE) did not detect this new impurity RP-HPLC/MS determined the identity of the impurity SEC-HPLC confirmed the impurity was not inter-particle aggregates Additionally, RP-HPLC-MS/MS (peptide mapping) identified increased
deamidation levels after the process change
Follow-up experiments confirmed/mapped the impurity was caused by a specific change in the process
Process change was removed because of greater analytical understanding of the product
Pfizer Proprietary
1. AAV Infection
Tissue Selective Infectivity of AAVSerotypes
Find a cell line that can be infected
Readout: Usually qPCR
2. Protein Expression
Tissue Specific Promoters
Find a cell line that results in promoter activity and protein expression
Readout: ELISA, western blot
3. Protein Activity
Activity Assays require successful infectivity and protein expression
Required for pivotal trial material
Readout: Depends on the protein (e.g., enzymatic activity)
4. Relative Potency
Activity relative to a reference material enables • lot to lot consistency• controls for assay
variability
Reference Material choice is critical
Readout: Relative Potency (e.g., 103%)
Robust potency assay(s) are critical for comparability
Multi-Component Potency Assays
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Optimal target cell line
Infect with virus encoding Collect conditioned medium
from infected cells Assess Activity
Single , All-inclusive Potency Assay Encompasses: Infectivity Expression of Gene Functional Activity
210% 55%
Cell-Based Assay: Relative Potency
100% Vs 50% mock potency100% Vs 200% mock potency
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DOEs: Optimizing Potency Assays
Cell Line Screeningo Target cell line with maximum promoter
activity driving Gene-of-Interesto Cell line with high infectivity rate
Viral Infectiono Cell density at harvesto Cell density for infectiono Cell passageo Medium conditions to maximize infection and
downstream expression of Gene-of-Interest
Activity measurement
o Time of harvest of conditioned mediumo Choice of assay for activity measurement
Good potency assay robustness and precision are critical for gaining meaningful data to assess comparability.
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Process-Related Impurity: Host Cell DNA
• Residual host cell DNA levels in AAV products across industry tend to exceed regulatory guidelines (10 ng/dose)– Necessary to have process understanding to control as much as possible
and support comparability
• Advanced characterization is key for process and product understanding– Why can’t DNA be purified beyond a certain level?
• qPCR is used to quantitate DNA levels during AAV downstream processing
• When AAV samples are treated with benzonase or DNase I prior to qPCR, we find that a percentage of the DNA is protected, potentially inside the AAV particles
– What is the size of the residual DNA?• Qualitative assessment can be performed by PCR and agarose gel
electrophoresis
• Approaches such as qPCR with nested primers, electrophoresis and other techniques are under evaluation for quantitation of DNA of different sizes
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Host Cell DNA Size Characterization
Human 18S rRNA gene (1870 bp)
Fixed Forward Primer
• Forward primer(s) & different reverse primers were designed to attempt to amplify different lengths of a host cell gene sequence in AAV samples.
• PCR is performed and PCR products are visualized on an agarose gel to determine if DNA of varying sizes is present.
• This qualitative assessment tells us that we have host cell DNA >200 bp.
• This DNA appears to be protected from DNase by the particle.
Different Reverse Primers
PCR products of different sizes visualized on a gel
Fixed Forward Primer
~150 bp ~300 bp ~630 bp
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Sedimentation Coefficient (S20,w)20 40 60 80 100 120 140
c(s
20,w
)
0.00
0.05
0.10
0.15
0.20
Advanced: Analytical Ultracentrifugation
Direct quantitation Less susceptible to impurity interferences
Correlates well to electron microscopy images Ability to distinguish full, partially full, and empty particles
However, difficult to make a QC release assay
Empty
FullPartial
Traditional: Spectrophotometry
Full : Empty Ratio
Improved Characterization: Full/Empty Ratio
Vector Particle Content:
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11,000x30,000x
10% Empty
69% “Fully Full”
Sedimentation Coefficient (S20,w)50 100 150 200
c(s 20
,w)
0.00
0.02
0.04
0.06
0.08
0.10
Empty Capsids
FilledCapsids
Transmission Electron Microscopy (TEM)
Analytical Ultracentrifugation (AUC)
Sample enriched for full particles
Improved Characterization: Full/Empty Ratio
• TEM– Size and shape consistent with expectations– Empty & full capsids distinguished
• AUC– Separation of empty & full capsids
• TEM consistent with AUCPfizer Proprietary
Sedimentation Coefficient (S20,w)50 100 150 200
c(s 20
,w)
0.00
0.05
0.10
0.15
0.20
15,000x 11,000x
Empty Capsids
Transmission Electron Microscopy (TEM)
Analytical Ultracentrifugation (AUC)• TEM– High % empty AAV capsids observed
• AUC– High % empty AAV capsids confirmed
• TEM consistent with AUC
Sample enriched for empty particles
Improved Characterization: Full/Empty Ratio
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• Introduction of assays with advanced capabilities improves comparability assessments and product understanding
• Robust release assays are necessary for process and product understanding
• Orthogonal assays advance product characterization and support validity and understanding of release assays
• Advanced product understanding could facilitate better critical quality attribute definition
Presentation Conclusion
Pfizer Proprietary
Acknowledgements: For These Data
Bioassay & Impurity
• Phoebe Baldus
• Herb Runnels
• Stephanie Botos
• Emilia Byrne
• Suzanne DeMarco
• Savita Sankar
• Kun Zhang
Mass Spec & Biophysical
• Tom Lerch
• Thomas Powers
• Caitlin Wappelhorst
Project Progression Line
• Amanda Werle
• Tatiana Shapkina
• John Amery
• Missy Anderson
• Svetlana Averianova
• Jasmina Fazlic
• Halyna Narepekha
• Rathna Nostra
Quality Control
• Tom Schomogy
• Amy St. Charles
Special Thanks As Sponsors of These GTx Projects
• Pfizer’s Rare Disease Research Unit and Bamboo Therapeutics, Inc.
– Jude Samulski
– Josh Grieger
– Michael Linden
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