Potency Assays for Adenoviral Vectors. Bryan T. Butman, Ph.D. Sr. Vice President, Vector Operations GenVec, Inc.

Transcription

1 Potency Assays for Adenoviral Vectors Bryan T. Butman, Ph.D. Sr. Vice President, Vector Operations GenVec, Inc.

2 Overview of Presentation Overview of GenVec s Adenovector platform technology Potency assay design and features Controlling variability of Adenovector potency assays Summary

3 Product Platform GenVec has developed an Adenoviral vector platform for localized production of target proteins Deletions of Adenoviral Early genes to assure nonreplication of vector in recipient Complete E1 and E4 deletions in adenovirus genome Establishment of 293-ORF6 production cell line to complement for missing viral domains E1 and E4-ORF6 complementation This platform is being employed in the development of therapeutic and vaccine products for human and animal health

4 GV11 Vector/Cell Line System Vector Adenovirus E1 E3 E4 GV11 E1 Expression Cassette E4 Spacer Complementing cell line 293-ORF6 cell line (GenVec) No RCA generated in any GV11 vector production No E4 transforming gene expression Stable, well-characterized, nononcogenic E1 E4

5 Vector-based Gene Delivery and Protein Expression

6 GenVec s Potency Assay Matrix Assays for assessment of potency for lot release and stability monitoring: Vector particle concentration Vector infectivity Transgene expression Bioactivity of expressed transgene protein

7 Comparison of PU Methods Spect. PU AE-HPLC PU (260 nm) AE-HPLC PU (Fluor) 1E+12 1E+11 1E+10 1E+09 1E+08 PU/ml

8 Spectrophotometric Assay (PU/ml) Based upon absorbance of UV light at 260 nm when vector particles are lysed by detergent (SDS) Extinction coefficient based upon Maisel, et al. Good correlation of assay value on Adenovirus Reference Material (ARM) Suitable for determining PU concentration in vector lots 2 e+11 PU/ml Used to assign value to Calibrator employed in HPLC- PU assay For vector lots < 2 e+11 PU/ml

9 Performance Characteristics of Spec-PU Assay Repeatability: 5% (between replicates within an assay) Intermediate Precision: 5% (between days and analysts) Linearity: r 2 > Back-calculated values < 1% error Robustness: 24 hr. delay (RT) in assay: 2% difference from T=0 Freeze/thaw (x3): 2% difference Varying SDS concentration (+/- 20%): 1% difference Specificity: Spiked CsCl does not interfere: < 2% difference from unspiked sample

10 Vector Particle Concentration by HPLC Method: Anion exchange chromatography Resin: Poros 50D (Applied Biosystems) Detection: UV or fluorescence Performance Characteristics: Accuracy: PVAC Controls < 10% variance from target Precision with 6 replicate injections: < 5% CV Intermediate Precision: < 5% CV between days and between analysts Linearity of Standard Curve: Rval Absence of drift within assay: <10% Robustness - System suitability criteria met for minor changes in: Column temperature, buffer ph, flow rate and elution buffer ionic strength

11 HPLC-PU: Chromatogram Norm min

12 Area Counts HPLC-PU: Standard Curve Fluorescence HPLC-PU Standard Curve Linearity 1 x 10 9 to 1 x PU/mL R = E E E E E E E+10 PU/mL

13 PU/mL HPLC-PU Assay in Stability Monitoring Accelerated Stability lot E E E E+10-20dC +5dC +25dC 0.0E Weeks

14 Vector Infectivity Assay Focus Forming Unit concentration (FFU/ml) Immunofluorescence assay based upon expression of early Adenoviral DNA binding protein (DBP) in nucleus of infected 293-ORF6 cells Positive control is run in each assay Reference FFU range established Used to establish system suitability FFU assay correlates well with IU/ml value on ARM Enables expression of PU:FFU ratio as an index of infectivity titer

15 Performance Characteristics of FFU Assay Repeatability: < 6% CV on replicates within assay Intermediate Precision: < 25% CV between analysts and days Specificity: No detection of heat-inactivated vector No detection in sample matrix (FFB alone) Adenovirus serotype 5 specificity Robustness: < 5% CV from cell line passages (passages 3 20) Approximately 25% CV from incubation time variance (24 +/- 2 hr) and immuno-reagent variance (different lots of antibody conjugate) Sample stability: Freeze/Thaw (x3): < 15% CV 24 hr RT hold: < 20%CV

16 FFU/mL FFU Assay in Stability Monitoring Accelerated Stability lot E E E E+08-20dC +5dC +25dC 1.0E Weeks

17 PU:FFU Ratio PU:FFU Ratio in Stability Monitoring oC +5oC +25oC Days

18 Transgene Expression Assays Host cells infected with Adenovector at fixed PU:cell ratio Transgene expressed during 24 to 48 hours in culture Phase 1/Phase 2 - Qualitative assessment of expression Protein: Western blot mrna: RT-qPCR Phase 2/Phase 3 - Quantification of transgene protein concentration TNF Commercial TNF- ELISA VEGF Commercial VEGF121 ELISA PEDF In-house PEDF ELISA Values calculated in terms of fg/cell or pg/cell Reference Standard run in parallel to control for inter-assay drift Results expressed as ratio of Test Article : Reference Standard

19 Calibration Curve for TNF-α ELISA

20 Performance Characteristics of TNF Expression ELISA System suitability for ELISA Ave. OD for blank 0.2 AU Ave. OD for top calibrator 1.0 AU Precision of calibrator replicates 10% CV Back-calculated values for calibrators 80 to 100% of target Linearity (r 2 ) of standard curve 0.98 System suitability for Controls TNFerade Reference Standard 500 pg/ml AdNull Negative Control below LLQ Acceptance criteria for sample TA:RS ratio: 0.53 to 2.25

21 TNFerade Lot Release Potency Assay Data Vector Lot PU/ml FFU/ml PU:FFU TNF TA:RS Number Ratio ELISA Ratio 1 4.6e e fg/cell TA 3.9 fg/cell RS 2 6.7e e fg/cell TA 2.5 fg/cell RS 3 3.3e e fg/cell TA 2.2 fg/cell RS 4 5.2e e fg/cell TA 1.5 fg/cell RS 5 6.9e e fg/cell TA 4.0 fg/cell RS 6 6.0e e fg/cell TA 2.9 fg/cell RS 7 5.7e e fg/cell TA 2.9 fg/cell RS 8 5.8e e fg/cell TA 9.2 fg/cell RS 9 5.6e e fg/cell TA 9.2 fg/cell RS

22 VEGF (% Control) Transgene Expression (VEGF) L L L +5 14H H H Months

23 Bioactivity Assay TNFerade - TNF-induced cell killing assay Target monolayer cells are sensitive to TNF- WEHI-13VAR cells Metabolic substrate (MTT) used as indicator Actively metabolizing cells take up MTT and enzymatically generate formazan dye, which can be read with a spectrophotometer Absence of signal = Cytotoxicity from TNF- Samples and Reference Standard run in triplicate (96 well) Standard curve generated for each in range of 0.1 to 25 pg/ml (based upon Transgene Expression ELISA) 4 parameter curve fit applied

24 TNFerade Bioactivity Assay 2 Plate Concentration y = ( (A - D)/(1 + (x/c)^b ) ) + D: A B C D R^2 Std (G-1083RS ( ): Concentration vs MeanValue) Test Sample ( (T=6) +23: Concentration vs Mea

25 Performance Characteristics for TNF Bioactivity Assay System suitability criteria apply to both RS and TA Lower asymptote value ( D ) 0.3 AU Dynamic range ( A-D ) > 1.0 AU Slope ( B ) in range of 1.0 to 2.0 Correlation coefficient (r 2 ) Signal ratio from Cell vs. No Cells Controls 8 Sample specification: Relative potency (TA:RS ratio of C-values from X-Axis) must be 75% to 125%

26 Controlling Variability in Adenovector Potency Assays Challenge: Adenovector potency assays are subject to significant intraand inter-assay variability Highly complex analyte More than 12 distinct proteins in the virus particle 32 KB of DNA Highly complex structure and function Requires cell infection, transgene expression and proper biological function of transgene

27 Inherent Variability in Cell-based Assays Potential variability through QC cell line passaging Metabolic state of cells can be highly variable at time of use Metabolic state of previous passage Age of culture post-passaging Degree of confluency at time of use Potential variability due to culture system reagents, materials and analyst Potential for edge effect in micro-plate assays

28 Addressing the Challenge (1) Establishment of QC cell banks for all assays Establishing criteria for QC cell line passaging and maintenance Control of maximum passage number Comprehensive Assay Development Report generated Establishment of detailed assay SOPs Clearly defined system suitability and assay acceptance criteria Product-Specific Reference Standard program Adenovectors are highly stable in the GenVec formulation when stored frozen (At least 5 years) Thorough characterization, stability monitoring and performance data trending for Reference Standards Use as infectivity controls to establish system suitability Provides comparator for transgene expression and biological activity assays

29 Addressing the Challenge (2) QC equipment qualification and calibration Well-trained QC Analysts Consistency and tracking of assay raw materials Begin assay validation evaluation early in clinical development Segregation of cell infection and protein quantification components in transgene expression assay validation Address cell infection optimization Control ELISA component

30 Summary GenVec has established a comprehensive potency assay matrix Controlling assay variability is essential Assay development, qualification and validation program is designed to keep pace with drug development GenVec s Adenovector products have a wellestablished, but maturing program for measurement of potency