TECHNOLOGY TRANSFER BATCH Flutcore’s meeting 12 th April 2016

PROCESS TRANSFERRED Fermentation at 10L at UCL Purification at IQUR Fermentation at 10L Purification COMPARABILITY

USP/DSP FIRST IMPRESIONS AT 3P The process seems not to be scalable at 100L bioreactor: DSP process uses only a few part of the biomass produced on 10 litres scale 2 SECs are not enough for purification. A different chromatographical step should be included IQUR’s loading (in volume) of the S1000 column is over the suggestion of the manufacturer 90 cm height for chromatographic resins (specially SEC) for industrial processes are too high. PROCESS TRANSFERRED

USP/DSP FIRST IMPRESIONS AT 3P Formulation of intermediate and final product not defined No stability data for final product Analytical criteria used for pooling fractions after the S1000 chromatography is based in non quantitative criteria List of IPCs to monitor the process considering a future should be updated and adapted PROCESS TRANSFERRED

USP TECHNICAL DISCUSSION Growth Kinetics

Growth Rate (LN OD) USP TECHNICAL DISCUSSION

Pellet Wet Weight USP TECHNICAL DISCUSSION

SDS-PAGE ANALYSIS

CONCLUSIONS Fermentation performance at UCL and 3P was similar USP TECHNICAL DISCUSSION Improvements can be performed: Change induction time Improvement of the fed batch phase

PROCESS TRANSFERRED

DSP TECHNICAL DISCUSSION

Tech Transfer at UCL/IQURRun Lysis Lysis Buffer 50 mM MOPS, pH 7.5, 5 mM DTT, Protease Inhibitor (AEBSF) 2 mM Benzonase 5 u/mL Lysis Ratio0.5 g Wet Weight / mL Cell disruption Micronisation APV Gaulin Micron Lab40, 3 passes at 500bar 8ºC Cell disruptor PANDA 3 passes at 500bar 8ºC Solubilization BufferTriton 0.1%, Conditions1 hour, 4ºC1 hour, 4ºC, stirring Clarification Centrifugation20000g, 30min, 4ºC15800g, 40min, 4ºC Dilution Buffer and ratio20 mM Tris, 10 mM EDTA, 2M Urea Ratio1:2 Filtration Size µm TFF concentration Cut off750KDa Permeate flux30LMH TMP6.0 psi Concentration fold40 Filtration Size 0.1 µm0.2 µm (5-8 mL per filter) The 0.1 µm filter clogs with less than 3 mL.

Tech Transfer at UCL/IQURRun SEC CL-4B Column volume490mL Load capacity5%5 % (24.4 mL) BufferTris 20 mM, EDTA 5 mM, Urea 1 M, pH 8.4 Flow rate22 cm/h ≈ 2mL/min22 cm/h=1.96mL/min StorageNo12 h at 4 °C SEC S-1000 Column volume1805 mL Load capacity2 %2% (36 mL) BufferTris 20 mM, EDTA 5 mM, pH 8.4 Flow rate15 cm/h ≈ 5mL/min15 cm/h = 4.91mL/min TFF concentration Cut off750KDa Permeate flux30LMH Final concentration mg/mL0 mg/mL Concentration fold 3 Vivaspin Concentration Cut off10KDa concentration mg/mL0 mg/mL Concentration fold 27 DSP TECHNICAL DISCUSSION

Chromatograms CL4B

Chromatograms S1000 DSP TECHNICAL DISCUSSION

Load (mL)24.4 Total protein (mg/mL)43.8 Total protein (mg) Elution (mL)61.2 Total protein (mg/mL)1.24 Total protein (mg)75.9 Yield (%)7% Load (mL)36.1 Total protein (mg/mL)1.24 Total protein (mg)44.76 Elution (mL)84 Total protein (mg/mL)LOD SEC CL4B SF-1000 Quantitative results

DSP TECHNICAL DISCUSSION SDS- PAGE CL4B

WESTERN – BLOT CL4B DSP TECHNICAL DISCUSSION

Silver stain of fraction of S1000

DSP TECHNICAL DISCUSSION Pool after S1000

Comparability Studies IQUR and 3P yield between the first and second SECs was different. 3P looses the material a) VLP was degradated in the hold time b) Unspecific and irreversible bound at S1000 occur. (3P used a new resin and Iqur used a resin with many runs previously performed) Then, some experiments were performed at IQUR to elucidate this issue

The effect of a holding step Chromatograms obtained by IQUR were similar to the previous but different from 3P CL4B S1000 Data provided by IQUR

S h Holding Data provided by IQUR

Western Blot and Silver Stain 12 hours holding time No holding time Holding time does not affect to the VLP purification Data provided by IQUR

The effect of new Resin CL4B chromatogram was similar CL4B Data provided by IQUR

Western and Silver stain of the 1 st S1000 Run Data provided by IQUR 1 st run with the S1000 showed similar effect that the one found by 3P

Western and Silver stain of the 3 rd S1000 Run Fresh (unused) S1000 non-specifically binds protein until a saturation level is achieved Data provided by IQUR

ANALYTICAL METHODS Method Method proposed by iQur Method description received Qualification report received Current SOP considered satisfactory Summary of Method Status 3P proposal for use of methods In process control test Release test Characterisation test SDS-PAGE for molecular weight and purity determination Yes No Purpose not defined – Purity and/or Identity and when to use - (IPC, DS, DP etc). Qualification not provided. Load amount not known. When to use Coomassie and silver not defined. Reportable results not defined. Reference is not appropriate. Yesyes Western BlotYes No Qualification not provided Difference in detection technologies. Question whether the colourmetric approach affects sensitivity and thence 1º and 2º antibody concentrations. Maybeyes Total protein (Bradford) Yes No When to use is not defined (IPC, bDS, DP) No qualification Probably inaccurate No controls used or assay acceptance criteria defined Relationship to number of VLPs or antigens is not defined YesYes*No

Method Method proposed by iQur Method description received Qualification report received Current SOP considered satisfactory Summary of Method Status 3P proposal for use of methods In process control test Release test Characterisation test HCP By ELISAYesNo--- (for ProC and ProV yes, but not routine) YesNo Residual DNA by qPCR YesNo--- (for ProC and ProV yes, but not routine) YesNo High performance size-exclusion chromatography (HPSEC) Yes No Purpose not defined – Purity and/or Identity and when to use - (IPC, DS, DP etc). Qualification not provided. No SST or acceptance criteria defined. Load amount not known Reference standard is not available. General lack of information. Yes OTHERS Dynamic light scattering (DLS) YesNo-- ProbablyYes ANALYTICAL METHODS

CONCLUSIONS USP/DSP TECHNICAL CONCLUSION The process is not scalable at 100L scale as present The harvest post-induction time on fermentation can be optimized The DSP steps use only a few part of the biomass produced on 10 litres scale 2 SECs are not enough for purification. A different chromatographical step should be included IQUR’s loading of the S1000 column is over the suggestion of the manufacturer. S1000 binds inexpecifically to the VLPs 3P and GE experts do not recommend 90 cm for S1000 and could impact future scaling up of the process

Absence of formulation for both intermediate and final product affect stability List of IPCs to monitor the process considering a future scale up are required USP/DSP TECHNICAL CONCLUSION 3P’s recommendations: Try using Sepharose based resins Try Capto 700 Resin Change the strategy of batch, fed batch phase and induction Change the final formulation and perform stability studies The project is still under development phase CONCLUSIONS

There is no reference standard available for VLP1 and VLP2. VLP1 and VLP2 have not been formulated, and the stability of VLP1 and VLP2 is not known. Furthermore, the stability of any reference standard would be questionable (even if frozen). The specifications are only partially defined. Many methods that would be expected are not provided yet. The application of the methods is generally unknown, and when they should be used for process monitoring, IPC, or final product release (bDS and DP). Analytical Requirements CONCLUSIONS