FLUTCORE 6M project meeting - PowerPoint Presentation

Slide1

FLUTCORE 6M project meeting

Pampaloma

April, 2016

Olotu

Ogonah

,

Ben

Blaha

,

Tarit MukhopadhyaySlide2

Last time from L

ondon

Transferred fermentation protocol from UCL to 3P

Based on Mixed Feed induction for 48hrs

Robust and

reproducable

for both VLP1 and VLP2.Slide3

Tech transferred to 3P

Initial fermentation for 20hr on glycerol

Followed by 48hrs induction on glycerol/methanol feedSlide4

Optical Density

Initially thought good

yeild

on biomass and product. Slide5

Initial recovery analysis of transferred process

Anti-core Blot

1 sec exposure

iQur

Process Run

5. 20K

pellet

4.

20K s/n

6. Diluted

s/n

1.

rHBc

(50ng)

2. Marker

3. Crude

lysate

1

2

3

4

5

6

1

2

3

4

5

6Slide6

CO

2

Traces (30L fermenters)Slide7

Harvest Biomass on Induction timeSlide8

Primary Recovery – Increase VLP release with increased pressure.

Current operating pressures at 500bar, 3 passes.

Questions possibility of increasing release with increased pressure.

Worry about co-impurity

Separation based on size and chargeSlide9

No homogenisation – sample peak at ~5.3umSlide10

Sample homogenised at 300bar, 1 passSlide11

Sample homogenised a 300 bar, 4 passes – creation of micronised debris below 100nmSlide12

Sample homogenised at

1200 bar

, 1 pass – assumes full disruption of the cellSlide13

Process Development of VLP recovery - FLUTCORE

Olotu Ogonah

Benjamin Blaha

Tarit Mukhopadhyay

Dept. of Biochemical EngineeringSlide14

UCL Remit

Commercial Fermentation and DSP process development.

Fermentation-

Almost complete. Further optimisation ongoing.

DSP

Primary recovery – still potential for higher yields by homogenisation optimisation.

Dual SEC process transferred to 3P

Final product contains a single major contaminant

Further purification difficult without use of additional orthogonal purification method.Slide15

Polishing Step -Hydrophobic Interaction Chromatography (HIC)

Products (VLP1 and VLP 2) can be purified by differential precipitation.

VLP recovery low.

Evaluate HIC as a final polishing step

Evaluation requires precise, high throughput assay.

Octet:

real-time, label-free analysis for determination of

concentration.Slide16

Octet: Bio-Layer Interferometry (BLI)

Binding

between a ligand immobilized on the biosensor tip surface and an

analyte

in solution produces an increase in optical thickness at the biosensor

tip.

This results in a wavelength

shift of the reflected light,

Δλ

which is a direct measure of the change in thickness of the biological layer.Slide17

Octet Quantification Assay outline

Target:

Differentiate between VLP and monomer

High throughput

Samples to be

assayed

with

zero sample

clean up.

Robust

High precision

Biosensor tip Rehydration

Antibody Loading

Antigen binding

Data Analysis*

Assay outline: 50 samples: ~ 2 – 4 hours.

*Quantification using initial slopes from adsorption isothermsSlide18

Loading Step: Anti-Mouse IgG Capture (AHC) Biosensors

•

Captures the IgG with a known orientation, maximizing activity

• Biosensor can be regenerated back to the

anti-mouse

Fc capture surface

•Compatible with buffer or cell culture media Slide19

Octet quantification process diagram

Load sensors in 20



g/ml 10E11 Mab

Assay a dilution series of purified HA2.3,(M2e)3

Construct a calibration curve or initial rate vs concentration

Repeat 6 times with regeneration after each assay.

Biosensor Regeneration

Biosensor tip Rehydration

Antibody Loading

Antigen binding

Biosensor Regeneration

Biosensor tip Rehydration

Antibody Loading

Antigen binding

Biosensor Regeneration

Biosensor tip Rehydration

Antibody Loading

Antigen binding

Biosensor Regeneration

Biosensor tip Rehydration

Antibody Loading

Antigen binding

Biosensor Regeneration

Biosensor tip Rehydration

Antibody Loading

Antigen binding

Biosensor Regeneration

Biosensor tip Rehydration

Antibody Loading

Antigen bindingSlide20

Impact of number of regeneration cycles on antibody

(10E11,

20.0



g/ml)

adsorption isotherm.

1

3

6Slide21

Adsorption isotherms for purified HA2.3,(M2e)3 Calibration curve

The

initial binding

rate is

proportional to the c

oncentration.

100



g/ml

0.78



g/mlSlide22

Evaluation of quantification assay precision

Probe regeneration not an issue.

R square = 0.961Slide23

Comparison between BCA and octet measured protein concentrations

VLP ID

mg/ml (BCA)

mg/ml

(octet)

HA2.3,(

M2e)

3

Reference



Tube 1: 22/10/15 KM71H pHe7 HA2.3,(M2e)

3

100g prep I

2

1.718

14%

Tube 2: 20/10/15 KM71H pHe7 HA2.3,(M2e)

3 30L fermentor prep II 0.50.53

-6%Tube 3: 11/11/15 KM71H pHe7 LAH.H3,K1 0.40.42-5%Tube 4: 13/10/15 KM71H pHe7 LAH.H3,K1 No Triton 0.250.0484%

Tube 5: 15/10/15 KM71H pHe7 K1,K1 0.250.160%Tube 6: 18/10/15 KM71H pHe7 HA2.3,(M2e)3 100g prep II 330%

Tube 6: 18/10/15 KM71H pHe7 HA2.3,(M2e)3 100g prep II 33.14-5%***Slide24

Octet assay Summary

Works for purified material.

VLP clone specific

Probe regeneration not an issue (reduced assay costs).

Range:100



g/ml - ~ 5 

g/ml.Slide25

Hydrophobic Interaction Chromatography:

Adding a second dimension to current VLP purification process.

The VLP purification process transferred to 3P contains sequential SEC purification steps.

Produces particles with a normal distribution centred around the putative VLP size.

A single major contaminant present.

Can be removed by (NH4)2SO4 precipitation, but with poor VLP recovery.

Suggests differences in levels

of hydrophobicity Slide26

Hydrophobic Interaction Chromatography (HIC) resin screening

Will use GE predictor (96 well) plates to screen multiple low (4) and high (4) hydrophobicity resins, at 2 pH and two salt types.

First experiments is to determine the

mobile phase

upper limit salt concentration which does

not cause precipitation or

denaturation of VLP;

i.e. defining the  stability

window

This limit may be pH dependent.Slide27

Impact of NaCl

concentration on HA2.3,(M2e)3 VLP solubility

Intact VLP was re-suspended (@100



g/ml) in buffer (either

50

mM

Mops, pH 7.5 or 20

mM

Tris

, pH

8.5) in the presence of increasing concentrations of

NaCl

(0.0

mM – 2.0 M).Assay on octetSlide28

Impact of salt type on VLP binding in 20mM

Tris

, pH 8.5

100

mM

naCl

50

m

M

N

aCl

0

mM

(NH4)2SO4

0

mM

NaCl50 mM (NH4)2SO4300 mM NaCl

300 mM (NH4)2SO4Slide29

Impact of salt type on VLP binding in 50

mM

Mops, pH 7.5

N

aCl

0

mM

0

mM

NaCl

(NH4)2SO4

NaCl

0

mM

Slide30

Summary

Octet quantification assay in operation.

Works with purified solutions – end product assay.

To Do: spike cell lysate to test precision in crude solutions.

Assay is VLP clone specific.

Purified VLP falls apart in the presence of salt (> 50

mM

) *.

Assays to be repeated.

Look at quantification in semi crude solutions –

Spiked solutionsSlide31

Octet assay optimisation: impact of antigen, purity, and Mab on assay responseSlide32

VLP2 Octet assay optimisation : impact of purity and Mab on assay response.Slide33

Core VLP Octet assay optimisation : impact of purity and Mab on assay response.Slide34

Summary

High throughput assay for

purified

VLP

is working (Lower limit > 2 g/ml).

Purified

HA2.3,(M2e)

3

VLP

1

appear to

falls apart in the presence of salt (> 50 mM)

*: Implications for HICAssay for pseudo-crude samples possible.Testing ongoing with real samples

Necessary for process optimisation