3 Identification of the Critical Process Parameters (CPP): parameters that impact process performance.
4 HomogenisationThe Lab40 is a validated scale down model to the Manufacturing scaleUse 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. # Homog. passpHPressure barDetergent %% solids (w/w)GFP yield flur. UnitsDNA mg/l47.5650.00.13.04159.924.18.0300.01.015.01586.629.030.69.03993.337.04453.346.18.51000.043.43973.359.623499.952.246.96186.6107.42066.623.531.53853.3122.49199.931.3959.916.42753.3102.93726.654.643.27106.624.5953.37.7226.65.34979.918.02208.44568.4938.92815.72959.14907.75951.7758.79099.12049.78303.1948.83078.4MODDE 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 rangeResponses: GFP concentration and DNA concentration.Create a model which describes process behaviour.NameAbbr.UnitsSettingsPrecisionHomogenisation passpass#2, 3, 40.05pH7.5, 8, 8.50.1pressurebar300 to 100010detergentTri%0.1 to 1% solids%_sol3 to 150.2
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 passes3% 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 Fluorescence6% loadMeOH 30°C (Lab40)Mixed 20°C (Lab40)MeOH 20°C (Lab40)MeOH 20°C (Soni.)48S IS IS IS I3 batches run under different conditions.Yields and quality (multiple bands on western) vary greatly with fermentation conditions.Initial High temp fermentation data indicated most of material was in the insoluble fraction. These assays were done with samples homogenised with 15% load cell paste.Repeating assays with 6% load during homogenisation ( instead of 15%) increases soluble fraction as seen by western and GFP.GFP assay appears to overestimates soluble fraction compared to western.GFP asssay measure free GLP and GFP attached to a VLP, hence amplified signal ?Need robust assays able to distinguish free from attached antigen (Octet). Use as measure of stability?21Western blots (AB – HepB core protein)
10 Impact of # of homogenisation passes on DNA levels High DNA contamination.Allowable DNA content for final formulated product ~ <10 ng/dose.Data suggests need for a DNA removal step.At small scale benzonase is used to control DNA but all smaples are kept at 4C, thus may explain high DNA.Irrespective of this benzonase will not be used at scale because of costs and we would have to prove that all traces of the benzonase are removed from the final product.DNA/ml in 15% ~ 5X amt in 3%, but 3% has 5 fold increase in volume. Therefore total DNA to be removed is higher in 3%.Also current 15% process has a 3 fold dilution step prior to TFF.4 passes
11 The story so far…….. Next….. TFF Akta Crossflow 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.FloculationPrecipitationAIEXFermentation3 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….. TFFAkta Crossflow
12 What is Tangental Flow Filtration (TFF)? TFF - Fluid continuously sweeps the membrane surface, recirculating the feed stream across the membrane.AdvantagesMinimizes 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-upTwo typesCassettes: High shear, High flux → Higher ∆PHollow Fibre: Low shear, lower flux than cassettes. Suitable for fouling feed streams.I have indicated that we use CFF for diafiltration and concentration. So what exactly is CFF?
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.CassettesSteep Flux vs TMP curveSystem 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.For predictable operation parameter values are restricted to the linear region of Flux vs TFF curve.Cassettes: The slopes of the curves are very steep; sensitive to perturbations in TMP. System unstable as it requires high permeate pressure to maintain modest tmp, and thus avoid fouling. Issues will multiply as retenate stream becomes more concentrated (viscous). Also, already high P will increase as concentration factor increases – perhaps becoming unacceptable.
14 Summary Homogenisation: TFF Next Steps ↑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.TFFCassettes: Sensitivity to perturbations in critical process parameters suggests not suitable for scale up.Early data suggests Hollow Fibre preferred route.Next StepsBegin fermentation optimisationComplete 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 stepInclude 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.