Gene Therapy and AAV: Advancing Analytical Characterization to Improve Product Understanding, Control and Comparability Exercises CMC Strategy Forum 17 July 2017 Herb Runnels, PhD Pfizer, BioTx Pharmaceutical Sciences, Analytical R&D Pfizer Proprietary
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Gene Therapy and AAV: Advancing Analytical Characterization to Improve Product Understanding, Control and Comparability Exercises
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
Pfizer Proprietary
• 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
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:
Pfizer Proprietary
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
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.
Pfizer Proprietary
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
Pfizer Proprietary
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