Applications involving the ViroCyt Virus Counter in the production of various recombinant proteins

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1 Applications involving the ViroCyt Virus Counter in the production of various recombinant proteins Chris Kemp Kempbio, Inc. Frederick, MD USA

2 Presentation Summary Kempbio, Inc. Transient Expression: BacMam BacMam Applications IgG, Influenza VLP, ebola Zaire-dTM How we came to embrace the ViroCyt 2100/3100 Conclusion

3 Kempbio, Inc Pegasus Ct. Suite P Frederick, MD USA

4 Kempbio, Inc. Small, Private Company with 24 years of experience working as a CRO for the Biopharmaceutical industry Core Expertise Recombinant Protein Expression for Preclinical Applications Baculovirus Mammalian Transient Transfection Mammalian BacMam Transduction Hybridoma and Stable Mammalian Cell Lines Baculovirus Production Virus Generation and Plaque Purification Contract Manufacturing of Baculovirus and BacMam Virus Stocks

5 Presentation Summary Kempbio, Inc. Transient Expression: BacMam BacMam Applications IgG, Influenza VLP, ebola Zaire-dTM How we came to embrace the ViroCyt 2100/3100 Conclusion

6 BacMam Invitrogen 2008 Mammalian CMV Promoter Express rproteins in a variety of mammalian cells using a baculovirus as the delivery vehicle

7 Second Generation Bacmam Vectors VSV-G Coat Protein (Panels C-G) on envelope of budded virus allows increased entry into cells Rick Boyce 2011

8 GFP BacMam HEK-293

9 BacMam Aggregation BacMam at 1 month BacMam at 6 months Titer drops after filtration

10 BacMam Storage & Recovery Cryopreservation Protocol Filter HTS Titer on ViroCyt Virus Counter To 36mL aliquot of virus, add 4mL of sterile glycerol Aliquot to 30 x 1mL cryovials Store at -80C

11 BacMam Storage & Recovery Recovery and Ranging Protocol Thaw vial of virus Set up 2 x 50mL shake flasks SF9 at 1x10e 6 cells/ml 20uL of thawed virus to one flask 200uL of thawed virus to second flask Incubate for 3 Days Filter small sample of each and titer using Virus Counter

12 Virus Recovery from Cryopreservation &"!!#$!.%."!!#$!'% '"!!#$!'% -"!!#$!'%,"!!#$!'% PFu/mL +"!!#$!'% *"!!#$!'% P1 20uL P1 200uL P1 )"!!#$!'% ("!!#$!'% &"!!#$!'%!"!!#$!!% &% (% )% *% +%,% -% '%.% &!% Virus Stock Number

13 BacMam Protocol CHO and HEK For 1L- 100L expression Cells at 1.5 to 2x10 6 cells per ml for HEK-293 Up to 4x10 6 cells per ml for CHO-S Generate BacMam virus in Sf9 cells If >10% culture volume concentrate virus Determine virus titer using ViroCyt 3100 to determine if need concentration Add cells and incubate at 37 C + 5% CO 2 for h. Add 10 mm sodium butyrate (required for CHO, optional for HEK) Media formulations that work with BacMam LTC: Freestyle-CHO, Freestyle-293 Expression Systems: ESF SFM, ESF 921

14 BacMam Concentration Hollow-fiber: 500kDa MWCO Centrifugation Aseptic process 43,000 x g 19,000 rpm SS minutes Resuspend pellet PBS Media

15 Presentation Summary Kempbio, Inc. Transient Expression: BacMam BacMam Applications IgG, Influenza VLP, ebola Zaire-dTM How we came to embrace the ViroCyt 2100/3100 Conclusion

16 Effect of Cell Density & MOI on rigg Yield Cell Density: A = 2x10 6 cells per ml B = 3x10 6 cells per ml C = 4x10 6 cells per ml CHO-S/CD-CHO MOI = 50 (25:25) mg Purified IgG per Liter BacMam MOI IgG B Bacmam MOI: Various CHO-S/CD-CHO 4x10 6 cells per ml 1L Shake-Flask MOI = 50 96h. PT Harvest Purified Yield = 150 mg/l

17 Humanized rigg: 10L Bioreactor SDS PAGE Gel Coomassie Stained Non-Reduced Culture Supernatant Serum-Free Freestyle Medium HEK-293/MOI = 50 24, 48, and 72h. Post-Transduction 10-Liter Stirred-Tank Bioreactor Purified Yield = 140 mg/l Total Yield: 1.4 Grams rigg

18 Purified Yield IgG HEK/CHO: PEI/BacMam IgG mg/l Purified HEK293: PEI CHO: PEI HEK293: BacMam CHO: BacMam IgG Production Number

19 Medigen 2014 BacMam H7N1 VLPs

20 BacMam H7N1 VLPs First Attempt Ratio of H7:N1:gag was 1:1:1 Fresh Virus Tittered using the ViroCyt 2100 MOI was 10:10:10 (30 Total) HEK-293 Shake-Flask 1L Harvested supernatant at 120h. PT Purified by centrifugation Analyzed by TEM

21 Medigen 2014 BacMam H7N1 VLPs

22 BacMam H7N1 VLPs Second Attempt Ratio of H7:N1:gag was 1:1:0.5 Fresh Virus Tittered using the ViroCyt 2100 MOI was 14:14:7 (35 Total) HEK-293 Shake-Flask 1L Harvested supernatant at 120h. PT Purified by centrifugation Analyzed by TEM

23 BacMam H7N1 VLPs Medigen 2014

24 Ebola Zaire Glycoprotein Mayinga Zaire Strain 1976 Trimeric transmembrane protein Heavily glycosylated Mucin domain Transmembrane domain deleted = dtm MW ~120 kda Expressed as secreted product in HEK-293

25 Zaire-dTM Vector Comparison PEI Vector pci Transient Expression Vector CMV Promoter HA-Tag Synthetic Gene Human Signal Sequence Transmembrane domain deleted Codon Optimized for Human Cells BacMam Vector Gateway Entry Vector Synthesized in standard cloning vector with ATT sites flanking ORF Bacmid transfected into Sf9 for BacMam generation CMV Promoter Transmembrane domain deleted Human Signal Sequence Codon Optimized for Human Cells

26 1-Liter Z-dTM PEI and BacMam HEK-293 Cells 1x1000 ml flasks Duplicate loadings 1.5x10 6 cells per ml Freestyle, serum-free PEI: 4:1 ratio with DNA PEI: 1 mg DNA per L PEI: Harvest 96h. PT BacMam: MOI = 50 BacMam: Harvest 72h. PT

27 Z-dTM Purification Scheme: 1-40-liters Q Capture Step 10mL Q- Sepaharose/L Batch Bind 16h. Pack Column Wash Elute using linear gradient mm NaCl at ph 7.0 Size-Exclusion Load concentrated Q-capture pool onto S-200 Sephacryl column Buffer = PBS Flow = 0.5 ml per min. Run SDS PAGE Pool ZdTM fractions Concentration and QC Concentrate using Centricon 10kDa MWCO Sterile Filtration Vialing Coomassie-stained SDS PAGE and Western Blot (anti- ZdTM

28 Z-dTM PEI: Q-Sepharose Q-Sepharose FF 20 mm Tris ph 7 Linear Gradient mm NaCl 5 ml Fractions SDS PAGE Coomassie-stained

29 Z-dTM BacMam: Q-Sepharose Q-Sepharose FF 20 mm Tris ph 7 Linear Gradient mm NaCl 5 ml Fractions SDS PAGE Coomassie-stained

30 Z-dTM PEI: S-200 SEC Sephacryl S /60 PBS ph ml per min. 3 ml Fractions SDS PAGE Coomassie-stained

31 Z-dTM BacMam: S-200 SEC Sephacryl S /60 PBS ph ml per min. 3 ml Fractions SDS PAGE Coomassie-stained

32 Z-dTM BacMam and PEI Final SDS PAGE PEI Yield = < 0.5 mg/l BacMam Yield = 4 mg/l SDS PAGE Coomassie-stained Reduced Western Blot Anti-ZdTM BCIP/NBT Color Development

33 Z-dTM BacMam and PEI Final Western Blot PEI Yield = < 0.5 mg/l BacMam Yield = 4 mg/l SDS PAGE Coomassie-stained Reduced Western Blot Anti-ZdTM BCIP/NBT Color Development

34 Presentation Summary Kempbio, Inc. Transient Expression: BacMam BacMam Applications IgG, Influenza VLP, ebola Zaire-dTM How we came to embrace the ViroCyt 2100/3100 Conclusion

35 Scatter Plot: Plaque Titer vs ViroCyt vp/ml titer 1.20E E+08 r 2 = 0.95 Slope = pfu/ml per vp/ml Y-intercept = -3.7x10 6 pfu/ml 8.00E+07 pfu/ml 6.00E E E E E E E E E+09 vp/ml (ViroCyt)

36 Virus Production based on Plaque and ViroCyt Titers Virus Particles per ml 7.00E E E E E E A 591 B 751 A 751 B MOI = x10 6 cells per ml SF900-III A = Plaque Titer B = ViroCyt Titer 1.00E E Time Post-Infection (hours)

37 Baculovirus in Supernatant of Stirred-tank Bioreactor 3.00E E+09 Virus Particles per ml 2.00E E E+09 Sf9 Cells MOI = 2.0 Cell Density = 2x10 6 cells per ml Titers by ViroCyt 2100 Each point = Mean of 3 titers 5.00E E Time Post-Infection (Hours)

38 Baculovirus Particles in Culture Supernatant 2.50E E+09 Virus Particles per ml 1.50E E+09 High Five Sf9 5.00E E Elapsed Time (h.)

39 Conclusions BacMam mediated transient transduction is a powerful tool for the expression of a variety of complex recombinant proteins at scale. Optimal protein expression levels require fresh virus and a knowledge of the titer of each virus stock utilized. IgG is produce best at high MOI s Viral Glycoproteins, Glycosidases and VLP s use lower MOI s The ViroCyt Virus Counter is the only methodology that satisfies our needs for rapid and reliable BacMam virus enumeration.

40 Thanks Organizers, I&L Biosystems France and ViroCyt Kempbio, Inc. April Birch Heather Allen Aubrey Harbaugh Kerrie Kenefick Pat Kemp Medigen (Infuenza VLP) Peter Pushko Irina Tretyakova

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