SOP Name/ Date Barry Sugarman Signature on file Name/ Date Beth Hutchins Signature on file Title: Adenovirus Reference Material : 1 of 14 Name/ Date Jonna Ellsworth Signature on file Name/ Date 1.0 PURPOSE To monitor the real-time stability of the when stored at -55ºC and at 20ºC over a five year period. 2.0 SCOPE The Working Group (ARMWG) oversaw the production of a purified reference material consisting of Adenovirus Type 5 Wild-Type in a 20 mm TRIS, 25 mm NaCl, 2.5% (w/v) Glycerol, ph 8.0 formulation. The reference material is vialed in small (1.0 ml) glass containers, each with a Teflon-coated rubber stopper and crimp seal. The fill volume was 0.5 ml and the target concentration was approximately 5 x 10 11 particles/ml. The was purified as a single bulk, filtered, divided into four large volume aliquots (approximately 600 ml each), and frozen. Each aliquot was thawed individually, filtered, and filled into glass vials, stoppered, and then frozen back at 80ºC. There were four different final filtration dates, resulting in four lots, each with a different date of manufacture. All four T=0 dates for -80ºC are within a 10 day period. The ARMWG will assign the official particle concentration and infectious titer to the. Per the instructions of the ARMWG, one lot, designated 001503, will be characterized fully. The other three lots will be partially characterized. All four lots will be monitored for stability at -55ºC and at 20ºC over a five-year period. The ultra-low temperature of -55ºC represents the storage conditions for the at the repository ATCC, i.e., -70ºC. The low temperature condition of 20ºC was selected to provide information for those customers of ATCC that inquire as to stability at 20ºC. The ARMWG does not intend that the be stored at this temperature. Further, the ARMWG has provided two SOPs that must be utilized to monitor the in addition to the other methods approved by the ARMWG described in Canji s long-term stability study proposal. 3.0 LIMITATIONS This protocol applies only to the produced under the auspices of the Working Group.
2 of 14 4.0 REASON FOR CHANGE The requirement to perform the ARMWG SOP for Infectious Titer, RD0021, Adenovirus 5 WT Reference Material Standard Operating Procedure for Determination of Infectious Titer in 293 Cells in a 96-Well Format, was dropped from time points in favor of use of the Canji infectious titer method, QC0049, Infectious Titer Assay for Adenovirus Preparations using and the NAS Titer Calculation. This change is made because the variability in the ARMWG SOP is large and the assay is very cumbersome. The reliability and consistency of performance of the Canji method is much better. 5.0 REFERENCES 5.1 Minutes of the Working Group, September 5, 2001. Discusses and approves the final version of the long-term stability proposal from Canji, Inc., including the number of containers to be analyzed per time point and temperature condition. 5.2 RFP 12.0, Canji, Inc. Proposal for Long-term Stability, Aug-30, 2001 revision 5.3 RD0021, Adenovirus 5 WT Reference Material Standard Operating Procedure for Determination of Infectious Titer in 293 Cells in a 96-Well Format 5.4 RD0020, Adenovirus 5 WT Reference Material Standard Operating Procedure for Determination of Particle Concentration via Spectrophotometric Analysis 5.5 QA0012, Handling Deviations 5.6 QC0024, Handling Out Of Specification Results 5.7 QC0028, Master Plan for Stability Studies 5.8 QC0008, Particle Concentration by HPLC Assay 5.9 QC0036, Spectrophotometric Analysis of Purified Adenovirus Preparations 5.10 QC0049, Infectious Titer Assay for Adenovirus Preparations using and the NAS Titer Calculation 5.11 QC0053, Particle Size Analysis of Purified Adenovirus Preparations by Photon Correlation Spectroscopy 5.12 Electron Microscopy Method, Linda Obenauer-Kutner, SPRI
3 of 14 6.0 DEFINITIONS 6.1 Date of Manufacture (Ref 21 CFR 610.50): For productions not subjected to official potency tests,... (5) the date of final sterile filtration of a bulk solution... (italics added). 6.2 Date of Transfer: The date on which vials stored at -55ºC are transferred to storage at -20ºC. 6.3 Time (T=0) Testing: Quality testing for T=0 should be initiated within 30 days of the date of manufacture of the product. 6.4 Month: For calculation of time points, one month is defined as 30.4 days (equals 365 days/12 months). 7.0 MATERIAL LOT INFORMATION 7.1 Manufacturer of Product: 7.1.1 Introgen Therapeutics, Inc., 2250 Holcombe Blvd., Houston, Texas 77030 7.2 Manufacturer of Excipient: 7.2.1 Introgen Therapeutics, Inc., 2250 Holcombe Blvd., Houston, Texas 77030 7.2.2 Ad5 WT Reference Material Excipient ( Excipient ): 20 mm TRIS, 25 mm NaCl, 2.5% Glycerol (w/v), ph 8.0 7.3 Product Lot Numbers subject to stability testing protocol: 7.3.1, ATCC Part No. 10-00023, Lot No. 001503 7.3.2, ATCC Part No. 10-00023, Lot No. 001504 7.3.3 ATCC Part No. 10-00023, Lot No. 001505 7.3.4 ATCC Part No. 10-00023, Lot No. 001506
4 of 14 7.4 Container/Closure 7.4.1 Container: Glass vial 7.5 Label 7.4.2 Closure: Teflon-coated butyl stopper with crimp seal 7.5.1 Containers for this study are labeled as follows: ATCC VR-1516 Adenovirus Type 5 Reference Material 10-0023 0.5 ml Research Use Only Lot # xxxxxx 8.0 STORAGE CONDITIONS 8.1 is stored upright in a closed storage box held frozen at two temperatures: 8.1.1 At -55ºC in an ultra-low temperature freezer 8.1.1.1 Freezer set point: 80ºC 8.1.1.2 Acceptable temperature range: 55ºC to 90ºC 8.1.2 At -20ºC in a low temperature, non-cycling freezer 8.1.2.1 Freezer set point: 20ºC 8.1.2.2 Acceptable temperature range: 10ºC to 25ºC 8.2 The temperatures of the ultra-low and low temperature freezers are monitored and access is restricted to authorized personnel.
5 of 14 9.0 TEST METHODS 9.1 Rationale 9.1.1 Since there is no single stability-indicating assay or parameter that profiles the stability characteristics of a recombinant adenovirus product, an array of distinct physical and biological parameters will be monitored. 9.1.2 Lot number 001503 will be monitored closely according to Attachments A and B. 9.1.3 Lot numbers 001504, 001505, and 001506 will be monitored more selectively per Attachment C. 9.2 Physical Characterization Test Methods 9.2.1 Particle Concentration OD 260nm/SDS 9.2.1.1 Particle concentration will be assessed using the procedure provided by the Working Group, Canji RD0020. This method determines the number of virus particles per unit volume based on the absorbance value at 260 nm in the presence of a lysing agent, 0.1% (w/v) sodium dodecyl sulfate (SDS). 9.2.2 Particle Concentration HPLC Assay 9.2.2.1 Particle concentration will also be determined via Canji Procedure QC0008, Particle Concentration by HPLC Assay. This method determines the number of intact viral particles per milliliter by comparing peak area against that generated by an internal standard after chromatography on an anion exchange resin. The chromatographic profile may also provide stability-indicating information since new peaks or a change in peak size or shape reflect a change in the amount of intact virions. 9.2.3 Virus Aggregation OD 320nm/260nm (Light Scatter) 9.2.3.1 Virus aggregation will be monitored using Canji Procedure QC0036, Spectrophotometric Analysis of Purified Adenovirus Preparations. This method is an indirect measure of the degree of aggregation and monitors the ratio of absorbance at OD 320nm to OD 260nm. 9.2.4 Virus Aggregation Particle Size Analysis via Photon Correlation Spectroscopy
6 of 14 9.2.4.1 Virus aggregation will be monitored using Canji Procedure QC0053, Particle Size Analysis of Purified Adenovirus Preparations by Photon Correlation Spectroscopy. In this method virus particles are illuminated by laser light and their vibration is detected by measuring the intensity fluctuations of the scattered light with a photomultiplier. Particle size is then calculated from the recorded scattered-light intensity by (1) computing the autocorrelation function (ACF) and (2) performing a unimodal particle size analysis on the ACF. The results are expressed as the mean particle size and polydispersity. This method is a direct but qualitative measure of aggregation. 9.2.5 Virus Aggregation Electron Microscopic Analysis 9.2.5.1 Virus aggregation will be monitored using a procedure developed at SPRI. This is a qualitative but direct assessment of virus aggregation. The method requires that the virus preparation be diluted and applied to coated EM grids, then analyzed using transmission scanning EM mode. 9.3 Biological Characterization Test Methods 9.3.1 Infectious Titer ARMWG SOP 9.3.1.1 Infectious titer will be determined using the procedure of the Working Group, Canji Procedure RD0021, on a periodic basis only. Infectious titer is determined in a 96-well format on 293 cells provided by the ARMWG. At day 10, cytopathic effect is assessed in each assay well. The number of positive wells and test article dilution information are used to calculate the infectious titer with a formula that incorporates a correction for diffusion of the adenovirus particle (NAS infectious titer). 9.3.2 Infectious Titer Infectious Titer Using and NAS Titer Calculation 9.3.2.1 Infectious titer will also be determined using Canji procedure QC0049, Infectious Titer Assay for Adenovirus Preparations using and the NAS Titer Calculation. This assay uses a direct immunofluorescence antibody conjugate to detect the presence of infectious adenovirus in D22-293 cells two days postinfection. Infected cells in the population are detected by immunofluorecence staining for Ad hexon using flow cytometry. The number of positive events and test article dilution information are used to calculate the infectious titer with a formula that
7 of 14 incorporates a correction for diffusion of the adenovirus particle (NAS infectious titer). 9.4 Other Characterization Test Methods 9.4.1 Container Integrity/Sterility 9.4.1.1 Container integrity will not be assessed directly. Instead sterility will be assessed at specified time points. Five containers will be sent for sterility testing per USP. Bacteriostasis/fungistasis testing is not required as the product has already been shown not to interfere in sterility assessments. 9.4.2 Optional methods 10.0 TESTING TIME POINTS 9.4.2.1 If signs of material deterioration are observed, other methods such as determination of ph, visual inspection, etc., may be utilized to further analyze the material. 10.1 Samples stored at -55ºC, Lot numbers 001503, 001504, 001505, and 001506 10.1.1 Samples will be analyzed by the methods enumerated in section 8.0 at the following time points, using the testing schedule in Attachment A for Lot number 001503 and in Attachment C for Lot numbers 001504, 001505, and 001506. The test schedule indicates which methods are used at which time points. Additional testing may be performed based on the availability of material. 10.1.2 Time (T=0), analyzed within 30 days of date of manufacture, i.e., date of final sterile filtration 10.1.3 6 months; analyzed ± 2 weeks of the 6 month anniversary date of Time 10.1.4 12 months, analyzed ± 4 weeks of the 12 month anniversary date of Time 10.1.5 18 months, analyzed ± 2 weeks of the 18 month anniversary date of Time 10.1.6 24 months, analyzed ± 4 weeks of the 24 month anniversary date of Time 10.1.7 36 months, analyzed ± 4 weeks of the 36 month anniversary date of Time
8 of 14 10.1.8 48 months, analyzed ± 4 weeks of the 48 month anniversary date of Time 10.1.9 60 months, analyzed ± 4 weeks of the 60 month anniversary date of Time 10.2 Samples stored at -20ºC, Lot number 001503 only 10.2.1 Samples will be analyzed by the methods enumerated in section 8.0 at the following time points, using the testing schedule in Attachment B. The test schedule indicates which methods are used at which time points. Time (T=0) will be established as the date on which the containers are placed into storage at -20ºC, i.e., the date of transfer. 10.2.2 12 months, analyzed ± 4 weeks of the 12 month anniversary date of Time 10.2.3 24 months, analyzed ± 4 weeks of the 24 month anniversary date of Time 10.2.4 36 months, analyzed ± 4 weeks of the 36 month anniversary date of Time 10.2.5 48 months, analyzed ± 4 weeks of the 48 month anniversary date of Time 10.2.6 60 months, analyzed ± 4 weeks of the 60 month anniversary date of Time 10.3 Attachments A, B, and C indicate the number of containers per test. With the exceptions noted in the attachment tables, all tests are conducted using individual containers. If necessary, other tests than those indicated as sharing containers may be performed by sharing material allocated for another assay. 10.4 Test articles will be thawed at room temperature. Material will be visually inspected once thaw is complete. Vials will be placed at 2-8 C or on ice after thaw if testing does not commence within 90 minutes of thaw. Material must be visually inspected again prior to testing. Testing must begin within 6 hours of thaw. If vial is placed at 2-8 C or on ice, the vial must be equilibrated to room temperature prior to beginning the assay. This can be accomplished by leaving the vial at room temperature for 15 minutes. 11.0 TIME POINT ACCEPTANCE CRITERIA AND DATA EVALUTION 11.1 Particle concentration OD 260nm/SDS
9 of 14 11.1.1 Report a single result per time point, averaging the value of n=4 dilutions, as per the ARMWG procedure. 11.1.2 If the value is greater than ± 20% of the ARMWG assigned label concentration, conduct an OOS investigation. If the value is determined to be valid, notify the ARMWG chairperson. 11.2 Particle concentration HPLC Assay 11.2.1 Report a single result per time point, averaging the value for n=2 containers, three measurements for each vial. 11.2.2 If the value is greater than ± 20% of the ARMWG assigned label concentration, conduct an OOS investigation. If the value is determined to be valid, notify the ARMWG chairperson. 11.3 Infectious Titer ARMWG SOP 11.3.1 Report a single result per time point, averaging the value for n=2 independent dilution series, as per the ARMWG procedure. 11.3.2 If the value is greater than ± one log of the ARMWG assigned label infectious titer, conduct an OOS investigation. If the value is determined to be valid, notify the ARMWG chairperson. 11.4 Infectious Titer - Infectious Titer Using and NAS Titer Calculation 11.4.1 Report a single result per time point, averaging the values for n=2 independent dilution series. 11.4.2 If the value is greater than ± one log of the T=0 data value, conduct an OOS investigation. If the value is determined to be valid, notify the ARMWG chairperson. 11.5 Virus Aggregation via OD 320nm/260nm (Light Scatter) 11.5.1 Report a single result per time point, averaging the value for a minimum of 2 measurements. 11.5.2 If the value is more than ± 30% of the T=0 data value, conduct an OOS investigation. If the value is determined to be valid, notify the ARMWG chairperson. 11.6 Virus Aggregation via Particle Size Analysis via Photon Correlation Spectroscopy
10 of 14 11.6.1 Report a single result and the distribution per time point, based on an n=1 reading. 11.6.2 If the data indicates a substantial change in aggregation profile, conduct an OOS investigation. If the data are determined to be valid, notify the ARMWG chairperson. 11.7 Virus Aggregation via Electron Microscopy 11.7.1 This is a relative, qualitative assessment made in comparison to the T=0 data value. Record information on the presence and type of aggregates, their apparent frequency and distribution of sizes. 11.7.2 Report a single result and distribution per time point, based on an n=1 reading. 11.7.3 If there are substantial changes in the aggregation profile, notify the ARMWG chairperson. 11.8 Container Integrity via Sterility Testing 11.8.1 Report the result for 5 individual containers per time point. 11.8.2 If any of the 5 containers does not pass, additional studies may be conducted to confirm the container closure integrity. Designs for additional studies must be approved by the ARMWG prior to initiation. 11.9 Data Evaluation and Incorporation into Study Report 11.9.1 For quantitative analyses, the mean values and standard deviations will be plotted as a function of time. Linear regression analysis will be performed for individual values at each time point to assess the data. The slope will be calculated and evaluated for statistical significance from a zero slope. 11.9.2 For qualitative analyses, statistical evaluation is not possible. However all observations will be summarized and profile changes reviewed as to conclusions drawn. 11.9.3 All data, both quantitative and qualitative, and statistical analyses will be summarized, tabulated, and presented graphically in the final study report. 12.0 DOCUMENTATION AND RESULTS REPORTING 12.1 Record keeping
11 of 14 12.1.1 All observations and data will be recorded in Canji/SPRI laboratory notebooks and/or on appropriate worksheets and forms. The following information must be recorded at each time point: 12.1.1.1 Time and date each container thawed 12.1.1.2 Temperature used to thaw each container 12.1.1.3 Visual inspection observations 12.1.1.3.1 After initial thaw 12.1.1.3.2 Prior to test initiation 12.1.1.4 Volume removed for each test 12.1.1.5 Time assay initiated 12.1.2 All data and assay reports will be reviewed by Quality Control and Quality Assurance to ensure accuracy and acceptability of the analysis. Deviations to this SOP and any established methods must be documented per Canji SOP QA0012, Handling Deviations. Other deviations should be recorded via memo or in notebooks as appropriate. 12.1.3 A written annual report will be filed with the Adenovirus Reference Material Working Group, summarizing all available data. 12.1.4 Once the five-year study is complete, a final report will be written. Copies of all data, files, and reports will be made available to the Adenovirus Reference Material Working Group. 12.1.5 Original files will be maintained by Canji, Inc., for the lifetime of the project, i.e., as long as is available.
12 of 14 13.0 ATTACHMENT A MONITORING STABILITY AT -55ºC; LOT NUMBER 001503 ONLY 13.1 Testing Schedule and Container Requirements Test & Time Points No. of Vials T = 0 6 mos 12 mos 18 mos 24 mos 36 mos 48 mos 60 mos OD 260nm/SDS 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Infectious Titer ARMWG SOP Infectious Titer cytometry Photon C Spectroscopy 2 2 If performed, ARMWG SOP ARMWG SOP If performed, If performed, If performed, If performed, If performed, 2 2 2 2 2 2 OD 320nm/260nm 1 1 nd 1 nd nd 1 1 1 Electron Microscopy 1 nd 1 nd 1 1 1 1 Sterility nd nd 5 nd nd 5 nd 5 Total No. of Vials 8 6 13 6 7 13 8 13 1 nd: Not Done.
13 of 14 14.0 ATTACHMENT B MONITORING STABILITY AT 20ºC; LOT NUMBER 001503 ONLY 14.1 Test Schedule and Container Requirements Test & Time Points No. of Vials 12 mos 24 mos 36 mos 48 mos 60 mos OD 260nm/SDS 2 2 2 2 2 2 2 2 2 2 Infectious Titer ARMWG SOP Infectious Titer cytometry Photon C Spectroscopy If performed, use same If performed, use same If performed, vials as If performed, vials as If performed, vials as 2 2 2 2 2 vials as vials as vials as OD 320nm/260nm 2 1 nd 2 2 2 Electron 1 1 1 1 1 Microscopy Sterility 5 nd 5 nd 5 Total No. of Vials 14 7 14 9 14 Note: T=0 data are established under 12.0. T=0 date for time point determination established as the date that containers are placed at 20ºC; i.e., the date of transfer. 1 nd: Not Done.
14 of 14 15.0 ATTACHMENT C MONITORING STABILITY AT -55ºC, LOT NUMBERS 001504, 001505, AND 001506 15.1 Test Schedule and Container Requirements Test & Time Points No. of Vials T = 0 12 mos 36 mos 60 mos OD 260nm/SDS 2 2 2 2 2 2 2 2 Infectious Titer ARMWG SOP Infectious Titer Photon C Spectroscopy 2 nd nd nd ARMWG SOP 2 2 2 vials as vials as vials as OD 320nm/260nm 1 1 1 1 Electron 1 1 1 1 Microscopy Sterility 1 nd nd nd nd Total No. of Vials 14 7 9 14 1 nd: Not Done.