Optimisation of the Primary Purification Steps of a VLP production process

1. Optimisation of the Primary PurificationSteps of a VLP production process Olotu Ogonah Benjamin Blaha Tarit Mukhopadhyay

2. VLP Process Flow Chart – early phases

3. Identification of the Critical Process Parameters (CPP): parameters that impact process performance.

4. Homogenisation • The Lab40 is a validated scale down model to the Manufacturing scale • Use process knowledge and expertise to suggest critical process parameters (CPP): parameter which impact process behaviour (response) or critical product quality attributes (CQA). • Will confirm through model building. • In these experiments the responses evaluated will be selected for their potential impact on process efficiency and scalability; i.e. soluble vlp recovered and DNA concentration.

5. Homogenisation screening experiment design – model building. • MODDE software used to enhance the design of screening experiments. • These experiments will identify parameters which have a statistically significant impact on process efficiency. • Model VLP → GFP insert. • GFP concentration will be used for a surrogate for VLP/core concentration. • Parameter range • Responses: GFP concentration and DNA concentration. • Create a model which describes process behaviour.

6. Actual vs predicted plots (DNA and GFP)

7. Graphical presentation of model terms: The scale and centred coefficients can be used to evaluate the significance of the model terms.

8. Impact of homogenisation pass # on GFP yield 3 passes • 3% load →1.74 X (V/V) than 15% loading. • 3% load →8.75 X (g/g) than 15% loading. • Re-evaluation of initial fermentation data reveal low vlp yield was an artefact of the homogenisation conditions used. • Reducing loading from 15%→ 6% solids increases soluble VLP → ~ 55%) 4 passes

9. Inter-fermentation analysis: VLPs Mixed 20°C (Lab40) MeOH 20°C (Lab40) MeOH 30°C (Lab40) MeOH 20°C (Soni.) 6% load 48 Fluorescence S I S I S I S I 21 Western blots (AB – HepB core protein)

10. Impact of # of homogenisation passes on DNA levels 3 passes • High DNA contamination. • Allowable DNA content for final formulated product ~ <10 ng/dose. • Data suggests need for a DNA removal step. 4 passes

11. The story so far…….. • Homogenisation: • ↑pH, ↑pass#, and ↑pressure increases vlp release. • Low % solids (3%) gives better vlp release (8 fold (wt./wt.) increase) compared to current protocol. • ↑ % Load, ↑homogenisation pass #, and ↑pressure increase total DNA released. • High genomic DNA contamination indicate DNA removal step will be necessary. • Floculation • Precipitation • AIEX • Fermentation • 3 batches run under different conditions. • Yields and quality (multiple bands on western) impacted by fermentation conditions. • Repeating assays with 6% load during homogenisation ( instead of 15%) reinforces impact of homogenisation conditions on yield. • GFP assay appears to overestimates soluble fraction compared to western. • Assay measure total (free and vlp attached) GFP, hence amplified signal ? • Need assays to distinguish between free and attached antigen (Octet ?). Use as measure of stability? Next….. TFF AktaCrossflow

12. What is TangentalFlow Filtration (TFF)? • TFF - Fluid continuously sweeps the membrane surface, recirculating the feed stream across the membrane. • Advantages • Minimizes clogging membrane pores (fouling) and promotes consistent, long-term productivity. • Units can be cleaned, stored, and re-used as needed; i.e. it is very cost effective. • Suitable for scale-up • Two types • Cassettes: High shear, High flux → Higher ∆P • Hollow Fibre: Low shear, lower flux than cassettes. Suitable for fouling feed streams.

13. Hollow Fibre vs Cassettes: Initial Flux vs TMP At large scale systems are run in constant flux mode in linear (non fouling) region of the flux vs TMP curve. Cassettes • Steep Flux vs TMP curve • System difficult to control at lower TMP because: • Flux sensitive to perturbations in TMP. • Requires high ∆P and permeate pressures to maintain low TMP. Hollow fibres: • Very flat flux vs TMP curve. • Easier to maintain steady state. • Lower  pressure required to achieve acceptable permeate flow. Will tolerate fouling streams.

14. Summary • Homogenisation: • ↑pH, ↑pass#, and ↑pressure increases GFP (and by implication vlp) release. • Low % solids (3%) gives better vlp release: 8 fold (wt./wt.) increase over current protocol. • ↑ % Load, ↑homogenisation pass #, and ↑pressure increase total DNA released. • High genomic DNA contamination indicate DNA removal step will be necessary. • TFF • Cassettes: Sensitivity to perturbations in critical process parameters suggests not suitable for scale up. • Early data suggests Hollow Fibre preferred route. • Next Steps • Begin fermentation optimisation • Complete parameter screening/optimisation of TFF step. • Evaluate additional dead end filtration step as a replacement for centrifugation during primary clarification of homogenate. • Look at DNA removal step • Include Quality assays in evaluation of CPPs. • OCTET – biacore without the flow! Kinetic and concentration data. • TEM of vlp. • Define Critical Attributes (CQA) ?

15. Revised VLP Process Flow Chart – What the future may look like.

16. END

17. Identification of the Critical Process Parameters (CPP): parameters that impact process performance.

18. Summary of model statistics

19. Impact of Filter Type on ∆P at initial conditions Note: as concentration factor increases P will also increase