1. AEX & Purification

2. Purification update A reproducible method for VLP purification has been developed which routinely produces material of 80% plus purity However, VLP morphology is not homogenous suggesting some large core- containing complexes are formed It is possible that these complexes only form transiently so the time of harvest may need optimisation A final “clean-up” step may be required Sucrose gradients have been used to generate material for in vivo studies but these are not GMP scaleable A combination of SEC and AEX would be ideal but has proven problematic Finally, we need to quantify both purity and morphology on a routine basis and so a matrix of data has been proposed

3. Optimal harvest time

4. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP UCL fermenter Runs assembly test Samples from UCL of fermenter runs 16 (K1,K1 NB1 Harvest sample only) and 20 (HA2.3,(M2e) 3 C17-19S.SP) Samples 1 pre-induction sample. 3 samples taken over the induction period at various times. 1 Harvested cell paste sample. 2ml each sample run on CL4B XK16/20 in 20mM Tris Ph8.4, 5mM EDTA, 1M Urea

5. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP UCL fermenter Runs assembly test CL4B. 73.5hrsHarvest 75.25hrs K1,K1 Harvest Time 0 4.58hrs 26.9hrs

6. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP UCL fermenter Runs assembly test CL4B. Anti-core blot rHBc (50ng) Marker #13 #4#6 #7 #8#9 #10 #11 #5 #12 rHBc (50ng) Marker #13 #4#6 #7 #8#9 #10 #11 #5 #12 rHBc (50ng) Marker #13 #4#6 #7 #8#9 #10 #11 #5 #12 rHBc (50ng) Marker #13 #4#6 #7 #8#9 #10 #11 #5 #12 rHBc (50ng) Marker #13 #4#6 #7 #8#9 #10 #11 #5 #12 Time 0 4.58hrs 26.9hrs 73.5hrsK1,K1 Harvest rHBc (50ng) Marker #13 #4#6 #7 #8#9 #10 #11 #5 #12 Harvest 75.25hrs 1 min exposure 1 sec exposure1 min exposure VLP

7. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP UCL fermenter Runs assembly test CL4B. Silver stain of peak Void volume fraction (#4) from each run These data suggest that optimal expression is found around 24hrs post- induction This agrees with shake flask fermentation Expression decreases after this time Vaccine candidate yeast can be grown in fermenters

8. Sucrose gradients

9. Purification of VLPs from CL4B-S1000 columns by sucrose gradient VLP samples (all from standard CL4B – S1000 column preps) KM71H pHe7 K1,K1, 1.4mg/ml. BL21 coHe7 HA2.3,e, 0.8mg/ml. BL21 coHe7 HA2.9,e, 0.7mg/ml. 0.5ml each loaded onto 10 – 60% sucrose gradients (13ml) in 20mM Tris pH8.4, 5mM EDTA. Centrifugation conditions: Beckman SW40 Ti rotor 30,000 rpm3hrs4 o C RCF (avg) 113652 RCF (max)160070 k-Factor 244.1 0.5ml fractions collected from bottom.

10. Location of core in gradients by dot and western blot “Goldilocks” VLP region

11. Summary of results The presence of sucrose, particularly in early samples negatively impacted TEM analysis due to low adherence of proteins to the grids and poor staining. PHe7K1K1 VLPs increased in numbers from fractions 14 to 18. However, the lower numbers of VLPs in the earlier fractions may have been due to higher concentrations of sucrose (up to 4 %) in these diluted samples. Further dilution is unlikely to improve analysis as the resulting concentration of VLPs would be too low for TEM analysis. For the E.coli expressed VLPs, no VLPs were seen in fraction numbers lower than 16. Again, this is most likely due to the high concentration of sucrose in these fractions. BL21 CoHe7HA2.3: Large VLP assemblies were seen in fraction 16 with increasing debris present in later fractions. BL21 CoHe7HA2.9: VLPs were only seen in fraction 18 but these tended to be within aggregates or clumps.

12. YEAST produced VLPs passed over sucrose gradient post FPLC: SDS-PAGE/Western Blot of fractions. Fractions pooled for each construct and sucrose concentration reduced from ~40% to <1% by dilution and re-concentration on 10kDa cut off spin filters. Fractions of KM71H pHe7 HA2.3,(M2e) 3 C17.19S.SP also pooled and sucrose reduced to <1% in same way. Final concentration and volumes. VLP pool (#): 1.0mg/ml. 1.5ml. Total yield 1.5mg Dirty pool (#):2.3 mg/ml. 1.5ml. Total yield 3.4mg

13. pHe7 HA2.3,(M2e) 3 C17,19S Sucrose gradient purified VLP Goldilocks VLP regionThree bears VLP region Sucrose cushions can be used to improve the homogeneity of yeast VLP morphology This process is not perfect but should allow in vivo testing to continue

14. AEX development -AEX polish of SEC purified VLP

15. KM71H K1,K1 VLP AEX on CIM-DEAE Input run directly through UV detector before loading onto CIM- DEAE All protein bound but nothing eluted by salt gradient or 1M NaOH ml mAU W a s t e ^^ 3 8 3 7 3 6 3 5 3 4 ( C o m p l e t e d ) > E n d _ B l o c k ( I s s u e d ) ( P r o c e s s i n g ) ( C o m p l e t e d ) > E n d _ B l o c k ( I s s u e d ) ( P r o c e s s i n g ) ( C o m p l e t e d ) > E n d _ B l o c k ( I s s u e d ) ( P r o c e s s i n g ) ( C o m p l e t e d ) > E n d _ B l o c k ( I s s u e d ) ( P r o c e s s i n g ) ( C o m p l e t e d ) > Using prep from high copy clone. VLP positive fractions from S1000 (41-50) concentrated to 0.58mg/ml. Run on CIMmultus QA-1 monolith column (1ml column) Buffer A – 20mM Tris pH 8.4, 5mM EDTA Buffer B – 20 mM Tris pH 8.4, 5mM EDTA, 1M NaCl Final wash – 1M NaOH, 1M NaCl 2ml sample loaded Method run (based on column volumes) Column pre-equilibration (5 CV Buffer A) Sample load (2ml) Column wash (5 CV Buffer A) Elution (10 CV 0 – 50% linear gradient Buffer B. Step to 100% Buffer B (10 CV) High salt wash (5 CV 100% Buffer B) Low salt wash (5 CV Buffer A) NaOH wash (5 CV 1M NaOH)

16. KM71H K1,K1 VLP AEX on CIM-DEAE CIM-DEAE regeneration protocol Wash 1M NaCl, 1M NaOH (20 CV) Hold over night in 1M NaCl, 1M NaOH Wash 1M NaCl, 1M NaOH (10 CV) Wash H 2 O (20 CV) Wash 30% 2-Propanol (20 CV) Wash H 2 O (20 CV) Hold O/N Protein is tightly bound to matrix, only partially released by over-night soak in NaOH. Most seems to be bound by hydrophobic interaction since released by 2- Propanol

17. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-QA Input run directly through UV detector before loading onto CIM- QA All protein bound but only 25% of OD 280 in input eluted by salt gradient or even after 1M NaOH, 1M NaCl. Therefore, protein lost on column. rHBc (50ng) Marker KM71H pHe7 HA2.3,(M2e) 3.SPC1719S High copy clone Fraction 18 Fraction 17 Fraction 16 VLP fractions desalted 1 sec exposure 3 hr exposure

18. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-QA CIM-QA acid wash regeneration protocol 20mM Tris pH 7.0, 5mM EDTA 50% Acetic acid 20mM Tris pH 7.0, 5mM EDTA 50% Acetic acid 20mM Tris pH 7.0, 5mM EDTA Missing protein eluted in 1 st acid wash Although protein levels are low, nucleic acid still appears to be bound to the column

19. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-QA CIM-QA Benzonase regeneration protocol 50mM Tris pH 7.4, 2mM MgCl 2, 0.1M NaCl 200u/ml Benzonase Hold over-night 37 o C (#) 50mM Tris pH 7.4, 2mM MgCl 2, 0.1M NaCl # CIM-QA acid wash regeneration protocol following Benzonase treatment 20mM Tris pH 7.0, 5mM EDTA 50% Acetic acid 20mM Tris pH 7.0, 5mM EDTA 50% Acetic acid 20mM Tris pH 7.0, 5mM EDTA Post Benzonase treatment acid wash elutes protein (small amount) and further nucleic acid

20. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-QA at pH 7.0 Input run directly through UV detector before loading onto CIM-QA Nucleic acid content not significantly reduced by AmS0 4 ppt All protein bound No OD 280 in input eluted by salt gradient VLP precipitated by 40% AmSO 4 in attempt to reduce nucleic acid content

21. KM71H VLP stability at lower pH Intention to use CIM-DEAE to purify yeast VLPs at lower pH. To allow this SEC purified VLPs (stored in 20mM Tris, 5mM EDTA pH 8.4) were buffer exchanged to 20mM Tris pH 7.0 or 50mM Na-Citrate buffer pH 6.0 using CL-4B (XK16/20). VLPs are expected to elute in void volume. Exchanging K1,K1 to pH 6.0 retains integrity of VLPs as shown by elution in expected region. But Exchanging HA2.3,(M2e)3.C17.19S.SP VLP to pH 6.0 appears to cause VLPs to dissociate to constituent units. Exchanging HA2.3,(M2e)3C17.19S.SP VLP to pH 7.0 appears to be causing an intermediate level of instability. A secondary observation is that Nucleic acid also moves to the small molecule region of the column. This provides further evidence that the nucleic acid present in the VLP region is there due to it being part off or attached to VLPs.

22. KM71H VLP stability at lower pH K1,K1 pH 8.4 → 6.0 Fractions from Void peak held at pH 6.0 for 72 hrs and then re- run on CL4B Shows that K1,K1 is also unstable at low pH over extended periods

23. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-DEAE pH 7-4 Step Gradient

24. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-DEAE pH 7-4 1M NaCl Step Gradient Core eluted at pH7 1M NaCl is lost on concentration – suggesting aggregation

25. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP AEX on CIM-DEAE pH 7-4 1M NaCl Step Gradient pH 7 0-1M NaCl gradient Step pH 4 for 5 CV Re-ran pH 7 0-1M NaCl gradient

26. AEX development -AEX on lysate

27. Fraction 3 Fraction 4 Fraction 18 Fraction 19 Fraction 16 Fraction 15 rHBc (50ng) Marker Input Fraction 6 Fraction 17 Fraction 20 Note – Only detectable core signal is from fraction at end of sample loading period. Indicating that at this point column had become saturated in ability to bind core. Coomassie stain confirms this for other proteins also. 5 sec exposure 10 min exposure KM71H HA2.3,(M2e) 3 C17-19S.SP VLP lysate without urea. AEX on CIM-DEAE 1M NaCl gradient pH 8.4

28. KM71H HA2.3,(M2e) 3 C17-19S.SP VLP lysate in 1M urea. AEX on CIM-DEAE 1M NaCl gradient pH 8.4 Fraction 3 Fraction 4 Fraction 19 Fraction 20 Fraction 17 Fraction 16 rHBc (50ng) Marker Input Fraction 6 Fraction 18 Fraction 23 Note – loaded at half total protein load compared to previous sample. No apparent breakthrough of core, coomassie indicates breakthrough of other proteins. Some core signal detected in gradient at ~40% salt (0.4M NaCl) 10 min exposure Fraction 16 Fraction 17 Fraction 17 1M urea Fraction 18 1M urea Fraction 20 Fraction 19 rHBc (50ng) Marker Fraction 15 Fraction 18 Fraction 16 1M urea Fraction 19 1M urea 10 min exposure After concentration (4x), core signal was detectable. Sample without urea treatment eluting over 55 – 88% salt. Some eluting at 100%. Sample with urea treatment eluting at 40%.

29. KM71H K1,K1 VLP lysate without urea AEX on CIM-DEAE 1M NaCl gradient pH 8.4 Fraction 3 Fraction 4 Fraction 18 Fraction 20 Fraction 16 Fraction 15 rHBc (50ng) Marker Input Fraction 6 Fraction 17 Fraction 23 Significant core in all of flow-through indicating that column has saturated in ability to bind core (possibly higher core content of K1,K1 or lower affinity for DEAE of K1,K1.) Core signal eluting over 20 – 54% salt. At higher salt increase in high molecular weight aggregate seen. 1 sec exposure 10 min exposure

30. KM71H K1,K1 VLP lysate in 1M urea AEX on CIM-DEAE 1M NaCl gradient pH 8.4 Note – by end of this run back- pressure on had increased to a level at which column can no longer be used Fraction 3 Fraction 4 Fraction 20 Fraction 23 Fraction 17 Fraction 16 rHBc (50ng) Marker Input Fraction 6 Fraction 18 Fraction 24 1 sec exposure 10 min exposure Flow through exhibits strange behaviour, core signal decreasing, this cannot happen on AEX unless material is aggregating and fouling column – decrease in signal corresponds to rapid increase in back-pressure. Core signal eluting between 40 – 60% salt

31. KM71H pHe7 K1,K1 High Copy Number Clone lysate and lysate after 1M urea loaded onto CIMmultus DEAE AEX fractions desalted and 4 fold concentrated Fraction 16 Fraction 17 Fraction 17 1M urea Fraction 18 1M urea Fraction 23 Fraction 20 rHBc (50ng) Marker Fraction 15 Fraction 18 Fraction 16 1M urea Fraction 20 1M urea K1,K1 binds with lower affinity than HA2.3,(M2e) 3 C17-19S.SP Sample without urea elutes mainly ~ 20 - 50% salt With urea elutes over similar salt range. Urea does not seem to lower affinity of K1,K1 for DEAE 1 sec exposure 10 min exposure

32. KM71H HA2.3,(M2e) 3 C17-19S.SP and K1,K1 VLP Lysate with and without 1M urea AEX on CIM- DEAE Overall conclusions Both constructs bind DEAE K1,K1 has lower affinity than HA2.3,(M2e) 3 C17-19S.SP Urea treatment of HA2.3,(M2e) 3 C17-19S.SP reduces affinity to similar value as K1,K1 Overall recovery is low for both proteins – large amount of material appears to foul column Urea treated K1,K1 loading of column resulted in complete blockage of column – but it is possible that this was result of cumulative fouling rather than specific to K1,K1.

33. Is urea necessary for SEC preparation?

34. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP High Copy Clone VLP prep without urea Using High Copy clone (as defined by qPCR). Yeast induced without prior starvation period. 26g cells resuspended in 320ml lysis buffer (20mM Tris pH 8.4, 2mM AEBSF, 5mM DTT, 3000U Benzonase (93u/g)) lysed by pressure homogenisation 500 bar, 3 passes Soluble core extracted from crude lysate with 0.1% Triton X-100 for 1 hr and then centrifuged ~20k x g, 30mins, 4 o C Supernatant (320ml) diluted with 320ml 20mM Tris pH 8.4, 10mM EDTA, to give a final buffer of 20mM Tris pH 8.4, 5mM EDTA. Note – 1M Urea was omitted from this buffer. Vacuum filtered through 0.8µm, 0.45µm and 0.22µm membrane filters Final filtrate washed on 1MDa Pellcon TFF, reducing volume to 25ml. MDa retentate sonicated 2 x 10 sec at 50% power. Then filtered through 0.2µm syringe filter followed by 0.1µm syringe filter (filter washes with SDS buffer after use). Significant back pressure observed on passage through 0.2µm and 0.1µm filter. Stored overnight at 4 o C. Soincated 2 x 10 sec, Then re-filtered (0.2µm). VLPs purified using 2-step Automated AKTAPure program Filtrate applied to Sepharose 4B column (XK26/92) in 20mM Tris pH 8.4, 5mM EDTA, Note – no Urea in this stage. Void volume material (160-205ml corresponding to fractions 4 – 8 in previous method, 45ml) applied to Sephacryl S1000 column (XK50/92) in 20mM Tris pH 8.4, 5mM EDTA,

35. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP High Copy Clone VLP +/- urea: CL4B comparison to same scale Without urea void region is 70% of + Urea, Small protein regions are equal +/- urea Without urea more large size material is being removed on filters

36. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP High Copy Clone VLP prep without urea: CL4B Void on XK 50/92 S1000 0 100 0 200 No Urea 1M Urea

37. KM71H pHe7 HA2.3,(M2e) 3 C17-19S.SP High Copy Clone VLP prep without urea CL4B Void Run on S1000 XK50/92 rHBc (50ng) Marker #70 #38#44 #47 #50#53 #56 #59 #41 #65 rHBc (50ng) Marker #2 (Input) #8 #10 #18 #20 #23 #26 #29 #32 #35 rHBc (50ng) Marker #70 #38#44 #47 #50#53 #56 #59 #41 #65 rHBc (50ng) Marker #2 (Input) #8 #10 #18 #20 #23 #26 #29 #32#35 No Urea 1M Urea Increased degradation in absence of urea

38. Purification of VLPs from CL4B-S1000 columns by sucrose gradient rHBc (50ng) Marker #26 #7#12 #14 #16#18 #20 #22 #10 #24 rHBc (50ng) Marker #26 #7#12 #14 #16#18 #20 #22 #10 #24 KM71H pHe7 K1,K1 60% 10% KM71H pHe7 HA2.3,(M2e) 3 C17.19S.SP 1M Urea Yield = 0.04mg/g rHBc (50ng) Marker #26 #7#12 #14 #16#18 #20 #22 #10 #24 KM71H pHe7 HA2.3,(M2e) 3 C17.19S.SP No Urea Yield = 0.05mg/g Without Urea more core in ‘Goldilocks’ zone but more degradation – need EMs

39. Data reporting matrix

40. KM71H pHe7 HA2.3,M2e Twin High Copy Clone VLP prep Using High Copy (~) clone (as defined by qPCR). Yeast induced without prior starvation period. 26g cells resuspended in 320ml lysis buffer (20mM Tris pH 8.4, 2mM AEBSF, 5mM DTT, 3000U Benzonase (93u/g)) lysed by pressure homogenisation 500 bar, 3 passes Soluble core extracted from crude lysate with 0.1% Triton X-100 for 1 hr and then centrifuged ~20k x g, 30mins, 4 o C Supernatant (480ml) diluted with 320ml 20mM Tris pH 8.4, 10mM EDTA, 2M Urea to give a final buffer of 20mM Tris pH 8.4, 5mM EDTA 1M Urea. Vacuum filtered through 0.8µm, 0.45µm and 0.22µm membrane filters Final filtrate washed on 1MDa Pellicon TFF, reducing volume to 25ml. Unlike other higher copy clones no detectable back pressure detected. Then filtered through 0.2µm syringe filter followed by 0.1µm syringe filter (filter washes with SDS buffer after use). Significant back pressure observed on passage through 0.2µm and 0.1µm filter (2 x 0.1µm units used) Stored overnight at 4 o C. Sonicated 2 x 10 sec, Then re-filtered (0.2µm). VLPs purified using 2-step Automated AKTAPure program Filtrate applied to Sepharose 4B column (XK26/92) in 20mM Tris pH 8.4, 5mM EDTA, 1M Urea. Void volume material (160-205ml corresponding to fractions 4 – 8 in previous method, 45ml) applied to Sephacryl S1000 column (XK50/92) in 20mM Tris pH 8.4, 5mM EDTA,

41. KM71H pHe7 HA2.3,M2e Twin High Copy Clone VLP prep 20K s/n 20K pellet Dil s/n 0.1 filtrate Permetate MDa Sonicated CL4B Input 0.1 filter SDS wash MDa 0.2 filtrate rHBc (50ng) Marker Crude lysate 1 sec exposure TFF CL4B

42. KM71H pHe7 HA2.3,M2e Twin High Copy Clone VLP prep: CL4B Void on XK 50/92 S1000 Fractions pooled and concentrated to 10ml on 1MDa Pellicon. Then to 3ml on 10kDa spin filter. VLP Pool = #41 - 53, protein concentration 8mg/ml, total yield 24mg VLPVLP VLP

43. Purification of VLPs from CL4B-S1000 columns by sucrose gradient: SDS-PAGE/Western Blot of fractions. rHBc (50ng) Marker #26 #7#12 #14 #16 #18 #20 #22 #10 #24 60% 10% Fractions pooled and sucrose concentration reduced from ~40% to <1% by dilution and re-concentration on 10kDa cut off spin filters. Final concentration and volumes. VLP pool (#): 0.57mg/ml. 1.5ml. Total yield 0.86mg Dirty pool (#): 0.35mg/ml. 1.5ml. Total yield 0.53mg

44. Biochemistry summary data ParameterValueUnits Volume of culture1.8L Wet weight pellet26g Band visible on initial gel?Y(Y/N) TFFy = 4.7843x -0.5 R² = 0.9704 Equation AUC (S1000 fractions)27.5mAu*ml Purity (image analysis)20% Amount recovered24mg Yield0.09% Purity sucrose (image analysis)20% Amount recovered sucrose0.86mg Yield sucrose0.003% EM (image analysis)% VLP

45. Image analysis S1000 Sucrose 1648.719398.284337.0422202.772119.205 22115.2762130.5482327.8611584.2341052.234 32555.2052089.5981987.3552838.2251394.447 TC2494.9331792.4971778.3552164.1752136.912 51430.1542281.9622519.569190.2631617.054 6400.527133.3852362.125 71752.4972016.125400.335 81854.154851.2843009.832 Total13251.4711693.6814722.478979.6678319.852 % purity18.8%15.3%12.1%24.1%25.7%19.2% 11942.5981846.7192116.8912018.062 23274.9123911.0833326.6693846.79 TC2152.2552266.7191980.1841596.184 4757.456899.698728.163616.456 5477.335514.113382.213899.184 61873.9121464.0831118.548259.092 Total10478.4710902.429652.6689235.768 % Purity20.5%20.8%20.5%17.3%19.8%