Pampaloma April 2016 Olotu Ogonah Ben Blaha Tarit Mukhopadhyay Last time from L ondon Transferred fermentation protocol from UCL to 3P Based on Mixed Feed induction for 48hrs ID: 544145
Download Presentation The PPT/PDF document "FLUTCORE 6M project meeting" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
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