1. FLUTCORE 6M project meeting Pampaloma April, 2016 Olotu Ogonah, Ben Blaha, Tarit Mukhopadhyay

2. Last time from London Transferred fermentation protocol from UCL to 3P Based on Mixed Feed induction for 48hrs Robust and reproducable for both VLP1 and VLP2.

3. Tech transferred to 3P Initial fermentation for 20hr on glycerol Followed by 48hrs induction on glycerol/methanol feed

4. Optical Density Initially thought good yeild on biomass and product.

5. Initial recovery analysis of transferred process Anti-core Blot 1 sec exposure iQur Process Run 5. 20K pellet 4. 20K s/n 6. Diluted s/n 1. rHBc (50ng) 2. Marker 3. Crude lysate 123456 123456

6. CO 2 Traces (30L fermenters)

7. Harvest Biomass on Induction time

8. Primary Recovery – Increase VLP release with increased pressure. Current operating pressures at 500bar, 3 passes. Questions possibility of increasing release with increased pressure. –Worry about co-impurity –Separation based on size and charge

9. No homogenisation – sample peak at ~5.3um

10. Sample homogenised at 300bar, 1 pass

11. Sample homogenised a 300 bar, 4 passes – creation of micronised debris below 100nm

12. Sample homogenised at 1200 bar, 1 pass – assumes full disruption of the cell

13. Process Development of VLP recovery - FLUTCORE Olotu Ogonah Benjamin Blaha Tarit Mukhopadhyay Dept. of Biochemical Engineering

14. UCL Remit Commercial Fermentation and DSP process development. Fermentation- Almost complete. Further optimisation ongoing. DSP Primary recovery – still potential for higher yields by homogenisation optimisation. Dual SEC process transferred to 3P Final product contains a single major contaminant Further purification difficult without use of additional orthogonal purification method.

15. Polishing Step -Hydrophobic Interaction Chromatography (HIC) Products (VLP1 and VLP 2) can be purified by differential precipitation. –VLP recovery low. Evaluate HIC as a final polishing step –Evaluation requires precise, high throughput assay. Octet: real-time, label-free analysis for determination of concentration.

16. Octet: Bio-Layer Interferometry (BLI) Binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip. This results in a wavelength shift of the reflected light, Δλ which is a direct measure of the change in thickness of the biological layer.

17. Octet Quantification Assay outline Target: –Differentiate between VLP and monomer –High throughput –Samples to be assayed with zero sample clean up. –Robust –High precision Biosensor tip Rehydration Antibody Loading Antigen binding Data Analysis* Assay outline: 50 samples: ~ 2 – 4 hours. *Quantification using initial slopes from adsorption isotherms

18. Loading Step: Anti-Mouse IgG Capture (AHC) Biosensors Captures the IgG with a known orientation, maximizing activity Biosensor can be regenerated back to the anti-mouse Fc capture surface Compatible with buffer or cell culture media

19. Octet quantification process diagram Load sensors in 20  g/ml 10E11 Mab Assay a dilution series of purified HA2.3,(M2e)3 Construct a calibration curve or initial rate vs concentration Repeat 6 times with regeneration after each assay. Biosensor Regeneration Biosensor tip Rehydration Antibody Loading Antigen binding Biosensor Regeneration Biosensor tip Rehydration Antibody Loading Antigen binding Biosensor Regeneration Biosensor tip Rehydration Antibody Loading Antigen binding Biosensor Regeneration Biosensor tip Rehydration Antibody Loading Antigen binding Biosensor Regeneration Biosensor tip Rehydration Antibody Loading Antigen binding Biosensor Regeneration Biosensor tip Rehydration Antibody Loading Antigen binding

20. Impact of number of regeneration cycles on antibody (10E11, 20.0  g/ml) adsorption isotherm. 1 3 6

21. Adsorption isotherms for purified HA2.3,(M2e)3 Calibration curve The initial binding rate is proportional to the concentration. 100  g/ml 0.78  g/ml

22. Evaluation of quantification assay precision Probe regeneration not an issue. R square = 0.961

23. Comparison between BCA and octet measured protein concentrations VLP ID mg/ml (BCA) mg/ml (octet) HA2.3,(M2e) 3 Reference  Tube 1: 22/10/15 KM71H pHe7 HA2.3,(M2e) 3 100g prep I 21.71814% Tube 2: 20/10/15 KM71H pHe7 HA2.3,(M2e) 3 30L fermentor prep II 0.50.53-6% Tube 3: 11/11/15 KM71H pHe7 LAH.H3,K1 0.40.42-5% Tube 4: 13/10/15 KM71H pHe7 LAH.H3,K1 No Triton 0.250.0484% Tube 5: 15/10/15 KM71H pHe7 K1,K1 0.250.160% Tube 6: 18/10/15 KM71H pHe7 HA2.3,(M2e) 3 100g prep II 330% Tube 6: 18/10/15 KM71H pHe7 HA2.3,(M2e) 3 100g prep II 33.14-5% ******

24. Octet assay Summary Works for purified material. VLP clone specific Probe regeneration not an issue (reduced assay costs). Range:100  g/ml - ~ 5  g/ml.

25. Hydrophobic Interaction Chromatography: Adding a second dimension to current VLP purification process. The VLP purification process transferred to 3P contains sequential SEC purification steps. Produces particles with a normal distribution centred around the putative VLP size. A single major contaminant present. –Can be removed by (NH4)2SO4 precipitation, but with poor VLP recovery. Suggests differences in levels of hydrophobicity

26. Hydrophobic Interaction Chromatography (HIC) resin screening Will use GE predictor (96 well) plates to screen multiple low (4) and high (4) hydrophobicity resins, at 2 pH and two salt types. First experiments is to determine the mobile phase upper limit salt concentration which does not cause precipitation or denaturation of VLP; i.e. defining the stability window –This limit may be pH dependent.

27. Impact of NaCl concentration on HA2.3,(M2e)3 VLP solubility Intact VLP was re-suspended (@100  g/ml) in buffer (either 50 mM Mops, pH 7.5 or 20 mM Tris, pH 8.5) in the presence of increasing concentrations of NaCl (0.0 mM – 2.0 M). Assay on octet

28. Impact of salt type on VLP binding in 20mM Tris, pH 8.5 100 mM naCl 50 mM NaCl 0 mM (NH4)2SO4 0 mM NaCl 50 mM (NH4)2SO4 300 mM NaCl 300 mM (NH4)2SO4

29. Impact of salt type on VLP binding in 50 mM Mops, pH 7.5 NaCl 0 mM 0 mM NaCl (NH4)2SO4 NaCl 0 mM

30. Summary Octet quantification assay in operation. –Works with purified solutions – end product assay. –To Do: spike cell lysate to test precision in crude solutions. Assay is VLP clone specific. Purified VLP falls apart in the presence of salt (> 50 mM) *. –Assays to be repeated. Look at quantification in semi crude solutions – –Spiked solutions

31. Octet assay optimisation: impact of antigen, purity, and Mab on assay response

32. VLP2 Octet assay optimisation : impact of purity and Mab on assay response.

33. Core VLP Octet assay optimisation : impact of purity and Mab on assay response.

34. Summary High throughput assay for purified VLP is working (Lower limit > 2  g/ml). Purified HA2.3,(M2e) 3 VLP 1 appear to falls apart in the presence of salt (> 50 mM) *: Implications for HIC Assay for pseudo-crude samples possible. –Testing ongoing with real samples –Necessary for process optimisation