C043 iQur Project Update 03 rd Mar 2009

Upstream development Three constructs received: –pTAC28b-CoHo7e –pTAC28b-CoHo7SAg,e –pTAC28b-CoHo7e,HAVP1 Plasmids re-prepped to bulk up quantities All three transformed into: –DH1 –DH5 –BL21 Expression tests at 50 mL scale complete (analysis underway) LB broth replaced with animal free media

Downstream development Main focus of downstream development work –Lysis buffer composition –Cell disruption –Clarification –Concentration and buffer exchange

Lysis buffer composition –Four buffers plus control investigated Cell disruption buffer (control): –20 mM Tris, 125 mM NaCl, 10 mM EDTA, 5 % glycerol, 0.05 % PS-80 pH 8.5 Cell disruption buffer 1: –20 mM Tris, 125 mM NaCl, 10 mM EDTA, 0.5 % PS-80 pH 8.5 Cell disruption buffer 2: –20 mM Tris, 125 mM NaCl, 10 mM EDTA, 1 % PS-80 pH 8.5 Cell disruption buffer 3: –20 mM Tris, 125 mM NaCl, 10 mM EDTA, 1 % Triton X-100 pH 8.5

Lysis buffer composition Experimental procedure: –1 g Empty - sAg pellet per experiment –Re-suspend to 50 mL in appropriate lysis buffer –Sonicate 10 pulses 70 % power on ice –Centrifuge in Beckman Allegra, 3775 xg, 30 min at +4 ºC –Filter through 0.22 µm dead end filter –Decant supernatant

Western blot results – lysis buffer LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.05 µg/μL)4 3 Resuspended cells buffer (control)5 4 Post lysis buffer (control)5 5 Supernatant post centrifugation (control)5 6 Supernatant post filtration (control)5 7 Pellet buffer (control)2.5 8 Resuspended cells buffer (buffer 1)5 9 Post lysis buffer (buffer 1)5 10 Supernatant post centrifugation (buffer 1)5

Western blot results – lysis buffer LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.05 µg/μL)4 3 Supernatant post filtration (buffer 1)5 4 Pellet buffer (buffer 1)2.5 5 Resuspended cells buffer (buffer 2)5 6 Post lysis buffer (buffer 2)5 7 Supernatant post centrifugation (buffer 2)5 8 Supernatant post filtration (buffer 2)5 9 Pellet buffer (buffer 2)

Western blot results – lysis buffer LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.05 µg/μL)4 3 Resuspended cells buffer (buffer 3)5 4 Post lysis buffer (buffer 3)5 5 Supernatant post centrifugation (buffer 3)5 6 Supernatant post filtration (buffer 3)5 7 Pellet buffer (buffer 3)

Microfiltration development Cell harvest (25 Sep 2008) –Fermentation Start Volume = 26 L –Final Cell Pellet Wt. = g –Supernatant volume = L –Yield = 4.3 g/L Cell disruption –Niro Soavi Pony continuous high pressure cell disruptor –3 Barg –Samples taken after each pass

Clarification by microfiltration –3 Membranes trialled: 0.65 µm, 0.45 µm and 0.22 µm –Retentate volume reduced by 50 % (100 mL to 50 mL) –VLP passed through membrane with 5 dia-volumes (DV) buffer, cell debris retained in the retentate –Once microfiltration complete, permeate passed through 0.45 µm and/or 0.22 µm dead - end filter as appropriate (see flow diagrams) Microfiltration development

Process Flow - microfiltration

LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (0.5 µg/μL)2 3 Resuspended cells buffer10 4 Post lysis Homogenisation pass Post lysis Homogenisation pass Post lysis Homogenisation pass Permeate 0.45 post concentration (0 DV)10 8 Permeate 0.45 (1 DV)10 9 Permeate 0.45 (2 DV)10 Permeate 0.45 (3 DV)10 SDS-PAGE results - microfiltration

LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (0.5 µg/μL)2 3 Permeate 0.45 (4 DV)10 4 Permeate 0.45 (5 DV)10 5 Final retentate Post 0.22 µm NFF10 7 Permeate 0.65 post concentration (0 DV)10 8 Permeate 0.65 (1 DV)10 9 Permeate 0.65 (2 DV)10 Permeate 0.65 (3 DV)10

SDS-PAGE results - microfiltration LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (0.5 µg/μL)2 3 Permeate 0.65 (4 DV)10 4 Permeate 0.65 (5 DV)10 5 Final retentate Post 0.45 µm NFF Post 0.22 µm NFF

SDS-PAGE results - microfiltration LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (0.5 µg/μL)2 3 Post lysis Homogenisation pass Permeate 0.22 µm post concentration (0 DV) 20 5 Permeate 0.22 µm (1 DV)20 6 Permeate 0.22 µm (2 DV)20 7 Permeate 0.22 µm (3 DV)20 8 Permeate 0.22 µm (4 DV)20 9 Permeate 0.22 µm (5 DV)20 10 Final retentate 0.22 µm20

LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.05 µg/μL)4 3 Resuspended cells buffer5 4 Post lysis Homogenisation pass 15 5 Post lysis Homogenisation pass 25 6 Post lysis Homogenisation pass 35 7 Permeate 0.45 (5 DV)5 8 Final retentate Post 0.22 µm NFF Western blot results - microfiltration

LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.05 µg/μL)4 3 Permeate 0.65 (5 DV)5 4 Final retentate Post 0.45 µm NFF Post 0.22 µm NFF

Western blot results - microfiltration LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.05 µg/μL)4 3 Post lysis Homogenisation pass Permeate 0.22 µm post concentration (0 DV) 10 5 Permeate 0.22 µm (1 DV)10 6 Permeate 0.22 µm (2 DV)10 7 Permeate 0.22 µm (3 DV)10 8 Permeate 0.22 µm (4 DV)10 9 Permeate 0.22 µm (5 DV)10 Final retentate 0.22 µm10

Ultrafiltration development Concentration and buffer exchange by ultrafiltration –3 Membranes trialled: 1000 kDa, 500 kDa and 300 kDa nominal molecular weight cut-off –Microfiltration permeate (0.45 µm) concentrated x2 and diafiltered into x5 dia-volumes AEX load buffer

Process Flow - Ultrafiltration 0.45 µM Microfiltration Permeate Filtered through 0.22 µM UF/DF Using 1000 kDa Membrane 100 mL Start material Concentrated to 50 mL Diafiltered 5 DV UF/DF Using 500 kDa Membrane 100 mL Start material Concentrated to 50 mL Diafiltered 5 DV UF/DF Using 300 kDa Membrane 100 mL Start material Concentrated to 50 mL Diafiltered 5 DV

LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (1.5 µg/μL)2 3*UF Start material KDa membrane 0.22 µM Filtered Permeate KDa membrane UF/DF Retentate 1 DV KDa membrane UF/DF Retentate 2 DV KDa membrane UF/DF Retentate 3 DV KDa membrane UF/DF Retentate 4 DV KDa membrane UF/DF Retentate 5 DV KDa membrane UF/DF Permeate 10 SDS-PAGE results - ultrafiltration 1000 KDa Membrane 0.45 µM MF Permeate *Product from 0.45 µM microfiltration work

LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.5 µg/μL)4 3 *UF Start material KDa membrane 0.22 µM Filtered Permeate KDa membrane UF/DF Retentate 1 DV KDa membrane UF/DF Retentate 2 DV KDa membrane UF/DF Retentate 3 DV KDa membrane UF/DF Retentate 4 DV KDa membrane UF/DF Retentate 5 DV KDa membrane UF/DF Permeate 5 Western results – ultrafiltration 1000 KDa Membrane *Product from 0.45 µM microfiltration work

SDS-PAGE results - ultrafiltration 500 KDa Membrane LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (1.5 µg/μL)2 3*UF Start material KDa membrane UF/DF Retentate 1 DV KDa membrane UF/DF Retentate 2 DV KDa membrane UF/DF Retentate 3 DV KDa membrane UF/DF Retentate 4 DV KDa membrane UF/DF Retentate 5 DV KDa membrane UF/DF Permeate *Product from 0.45 µM microfiltration work

Western results – ultrafiltration 500 KDa Membrane LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.5 µg/μL)4 3*UF Start material KDa membrane UF/DF Retentate 1 DV KDa membrane UF/DF Retentate 2 DV KDa membrane UF/DF Retentate 3 DV KDa membrane UF/DF Retentate 4 DV KDa membrane UF/DF Retentate 5 DV KDa membrane UF/DF Permeate *Product from 0.45 µM microfiltration work

SDS-PAGE results - ultrafiltration 300 KDa Membrane LaneSample Vol. loaded (µL) 1 Benchmark Protein Ladder5 2 HBV core protein (1.5 µg/μL)2 3 *UF Start material KDa membrane UF/DF Retentate 1 DV KDa membrane UF/DF Retentate 2 DV KDa membrane UF/DF Retentate 3 DV KDa membrane UF/DF Retentate 4 DV KDa membrane UF/DF Retentate 5 DV KDa membrane UF/DF Permeate *Product from 0.45 µM microfiltration work

LaneSample Vol. loaded (µL) 1 Biotinylated protein ladder10 2 HBV core protein (0.5 µg/μL)4 3*UF Start material KDa membrane UF/DF Retentate 1 DV KDa membrane UF/DF Retentate 2 DV KDa membrane UF/DF Retentate 3 DV KDa membrane UF/DF Retentate 4 DV KDa membrane UF/DF Retentate 5 DV KDa membrane UF/DF Permeate Western results – ultrafiltration 300 KDa Membrane *Product from 0.45 µM microfiltration work

Conclusion Primary separations –Lysis buffer work no evidence for improved recovery Cell disruption – barg in continuous high pressure cell disruptor significantly increased release of product Microfiltration –0.65 µm membranes a possibility but process solution would still required dead-end filtration through both 0.45 and 0.22 µm capsule filters –Significant product retention observed using 0.22 filters therefore ruled out for further development 0.45 µm membranes selected Operating parameter scouting complete Ultrafiltration 1000 KDa MWCO membranes selected Operating parameter scouting complete