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AAPS Webinar
Sept 20th 2018
Jessica Dawson, Corinne Miller
Steven Degon, Sawako DeRosa, Brittany Tomascak
How to rescue a pH sensitive protein: Detergent viral inactivation and analytical quantitation of residual detergent
Detergent Viral Inactivation | 20.09.20182
Overview
Detergent Viral Inactivation
Detergent Viral Inactivation
History of Triton X-100
Requirements for new detergent options
Octyl-β-D Glucopyranoside (ODG)
Triton CG-110
ODG on viral inactivation
Fc-Fusion
Negative Impact of low pH viral hold on Fc-fusion molecule
Analysis of ODG with the Fc-fusion
Detergent Quantitation Method
Suggested WHO guidelines
Development of ODG detergent quantitation method with RP-HPLC and ELSD with minimal sample preparation
1
2
3
4
MilliporeSigma Emd Serono
Detergent Viral
Inactivation
4 Detergent Viral Inactivation | 20.09.2018
❑ Viral Clearance Studies
❑ Scaled down models for the process steps:
maintaining flow rates, column dimensions, column reuse,
binding capacity, filter size etc.
❑ Model viruses representing a wide range of
biophysical and structural characteristics are tested
Virus is spiked in at high titer at the start of the unit
operation. The virus clearance is determined for each step.
Log based reduction assay
Viral Clearance
5 Detergent Viral Inactivation | 20.09.2018
Viral Clearance
❑ 2-3 Orthogonal methods of viral clearance
Low pH hold – denatures enveloped viruses, easily performed after low pH elution off of Protein A but must have a low pH stable protein
Solvent/detergent – effective against enveloped virus with a lipid coat, detergent must be removed by process
Nanofiltration – removes viruses specifically by size exclusion
Chromatography – depends on the resin and buffers used during the process but can effectively remove all types of virus
~4-5 LRV ~3-4 LRV ~4-8 LRV
❑ The overall clearance should total ≥12-15 LRV
The clearance is express as a Log Reduction Value (LRV)
1 LRV = 10-fold reduction in virus (90% elimination)2 LRV = 100-fold reduction in virus (99% elimination)
~1-2 LRV ~1-2 LRV
ODG
7 Detergent Viral Inactivation | 20.09.2018
• Viral inactivation of 4-5 LRV
• Must fit into temperature and hold times of manufacturing purification process
• 4-25°C and < 2 hours of hold ; ideally no degradation of product on longer hold
time
• Minimal impact on protein quality
• Effective clearance of the detergent
Key Considerations in Evaluating Detergent
Cost and GMP Grade
Detergent Viral Inactivation | 20.09.20188
Next Step How does ODG function during a protein purification cascade?
ODG Provides required viral clearanceTemperature and time both fit with a protein purification process
ODG in an Fc-Fusion
protein purification
process
Detergent Viral Inactivation | 20.09.201810
In the Meantime…
The colleagues at EMD Serono were trying to purify this protein
Disulfide Bonds (inter)N-linked Glycosylation
The Foe : An Fc with N- and C-terminal fusions
Prone to aggregation under low pH, low salt, shear stress, and light stress
• N-terminal fusion
• Fc – N297Q mutation (no glycan)
• C-terminal fusion
Detergent Viral Inactivation | 20.09.201811
Impact of low pH hold on Fc-Fusion
Platform Viral Inactivation Low pH hold (30-60 minutes) for viral inactivation
Result: >30% increase in aggregateNO GO
Post - Protein A elution
77% monomer
42% monomer
Post- low pH hold
12 Detergent Viral Inactivation | 20.09.2018
Viral Inactivation: Detergent
❑ New process incorporates detergent inactivation before Protein A capture
AEX FT CEX AEX Detergent
Inactivation
Inactivation Removal
Enveloped VirusesEnveloped and Non Enveloped Viruses
Enveloped and Non Enveloped Viruses
Low pH Wet heat Virus Reduction
High pH Dry heat Chromatography
Solvent-detergent Gamma irradiation
Detergent UV-C
Adapted from KM Remington, BioProcess International 13(5):10-17
13 Detergent Viral Inactivation | 20.09.2018
Fc-Fusion: Detergent Viral Inactivation Study
Octyl D-glucopyranoside
Caprylic Acid
❑ Study performed by the Virology group at Millipore Sigma
❑ Detergent inactivation followed by PA purification to check for protein quality and recovery
Detergents to Evaluate
• Tween 80
• Triton CG-110 (mix of ODG and DDG)
• EcoSurf EH-9
• Octyl D-glucopyranoside
• Lauryldimethylamine-oxide(LDAO)
• Caprylic Acid (Octanoic Acid)*
* MilliporeSigma did not perform detergent study on these detergents
14 Detergent Viral Inactivation | 20.09.2018
Performed in DSP
❑ Viral Inactivation was performed on clarified harvest
❑ 5 detergents: Tween 80, Triton CG-110, EcoSurf EH-9, LDAO, ODG
❑ 6°C and 22°C
❑ 30min to 60min
❑ 0.5% to 1% Detergent Concentration
❑ VI supernatant was purified over PA pre packed column
❑ Recovery and Purity were determined for each run
Fc-Fusion
Detergent Screening
Detergent
Inactivation
15
Detergent Screening with Fc-Fusion
Screening Conditions:
➢ Virus: XMuLV
➢ Detergent conc.: 0.5% & 1%
➢ Incubation time: 30 min
➢ Temp.: 22oC
➢ All detergents with the exception of Tween® 80 provided complete inactivation within 30 minutes
Octyl and decyl β-D glucopyranoside
Detergent Viral Inactivation | 20.09.2018
16
mAb Recovery and Product Quality
➢ Triton™ CG-110 detergent excellent mAb recovery, minimal increased aggregation
➢ EcoSurf ™ EH-9 detergent acceptable mAb recovery, minimal increased aggregation
➢ LDAO detergent poor mAb recovery, increased aggregation
Detergent% mAb
Recovery%
Monomer
None 95.2 69.8
Triton™ CG-110 95.3 68.6
EcoSurf™ EH-9 93.8 66.4
LDAO 58.6 60.9
Detergent Viral Inactivation | 20.09.2018
17 Detergent Viral Inactivation | 20.09.2018
Detergent Screening Summary
Fc-Fusion
Detergent EffectivenessProtein Quality
Process Step Benefits Risks
LDAO +++Precipitation
Clarified Harvestor post Q
>4LRV, Eco-Friendly.Causes precipitation with Fc-Fusion. Low yield.
Ecosurf EH-9 +++ GoodClarified Harvestor Post Q
>4LRV, Eco-Friendly Not available in GMP grade
Triton CG-110 +++ GoodClarified Harvestor Post Q
>4LRV, Eco-FriendlyAssay available
Not available in GMP grade
Tween 80 +/- GoodClarified Harvestor Post Q
Readily available, Safe – Used in formulation buffer
Poor viral inactivation.Required extended exposure. No assay
Caprylic Acid?
Good Pre-clarificationReadily available. Could be used as flocculent as well
Low solubility. Unsure ofviral inactivation.Molecule dependent. Toxicity. No assay
Octyl-β-D-glucopyranoside(ODG)
+++ Okay Clarified Harvestor Post Q
Component of Triton CG-110.Assay available.
Cost $$$Need to test VIAlso not GMP grade
Slide by Steven Degon
X
X
Detergent Viral Inactivation | 20.09.201818
Next Step…
Can we quantitate and confirm detergent removal?
Detergent
Quantitation
Confirm
detergent
clearance
Detergent Viral Inactivation | 20.09.201820
Text slides and Textboxes
Objective of Detergent Quantitation Study
1 What is the acceptable limit of detergent?
WHO TRS No. 924 2004: Triton 3-25ppm.
2 Develop quantitation assay that can detect <25 ppm of ODG.
3 Develop protein removal method
Confirm that the protein purification process of Fc-Fusion removes detergent to < 25 ppm4
21 Detergent Viral Inactivation | 20.09.2018
▪ Separation method
Development of RP-HPLC method
Chromatography Selection
Octyl-D-glucopyranoside is a hydrophobic molecule
RP-HPLC commonly used mode of separation
Used Waters µBondapak C18, 3.9 mm x 300 mm, 10 µm, 125 Å
MpA: Water + 0.1% Formic Acid MpB: Acetonitrile
Separates based on hydrophobicity of analyte
Reverse phase columns have a nonpolar stationary phase consisting of hydrocarbon chains bonded to a silica gel base
The length of the hydrocarbon chains impacts the hydrophobicity of the stationary phaseLengths range from C3 to C18C18 would have the strongest hydrophobic interaction with an analyte
22 Detergent Viral Inactivation | 20.09.2018
▪ ELSD has three main components
Detergent Quantitation using ELSD
What is ELSD?
HPLC effluent is nebulized using inert nitrogen gas sparge
Nebulization Evaporation
Excess solvent is removed from analyte particles in heated tube
Light Scattering
Dried analytes enter light scattering cell and the scattering is measured by a photomultiplier tube
Images from http://www.sedere.com/the-low-temperature-evaporative-light-scattering-detector-lt-elsd/
Detergent Viral Inactivation | 20.09.201823
C18 RP-HPLC
HPLC Method Development
Nebulizer Temp
ELSD (evaporative light scattering
detector)
30-80°C
HPLC column temp
35-75°CInjection volume
20-100ul
Flow Rate
0.8-1.0mL/min
Evaporator Temp
30-80°C
Gas Flow
1.2-2.2
Selected parameter in white
Insert text
Detergent Viral Inactivation | 20.09.201824
Objective 2 – Detergent Quantitation Limit
Calibration Curve
min7.5 8 8.5 9 9.5 10 10.5 11
mV
0
100
200
300
400
100ppm
50ppm
25ppm
10ppm
8ppm
min8.4 8.6 8.8 9 9.2 9.4 9.6
mV
0
20
40
60
80
100
120
Quantitation to 8ppm!
Objective 2 met
y = 1,5857x + 0,0279R² = 0,999
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
0,00 0,50 1,00 1,50 2,00 2,50
Log
Are
a
Log Concentration
Calibration Curve8-100ppm ODG
Detergent Viral Inactivation | 20.09.201825
Spin concentrator
One step
Quick
Small volume
Impact on detergent quantitation unknown.
Solid Phase Extraction
Multi-step process
Time consuming
Material consumption?
Minimal interference with detergent quantitation
Objective 3 – Removal of protein
Protein Removal
Why do we need to remove protein?Protein will bind to C18 resin and potentially block detergent binding (impact on quantitation)
Will the detergent stick to the filter?
Detergent Viral Inactivation | 20.09.201826
Objective 3 – Removal of protein
Comparison of filtered and unfiltered sample
Sample Name Calc. Concentration Difference (ppm) % Error
100ppm Octyl-D 96.152.8 2.93
100ppm Octyl-D 1X Filtered 93.33
50ppm Octyl-D 51.812.6 4.97
50ppm Octyl-D 1X Filtered 49.24
25ppm Octyl-D 25.851 3.79
25ppm Octyl-D 1X Filtered 24.87
10ppm Octyl-D 9.810.5 5.37
10ppm Octyl-D 1X Filtered 9.28
8ppm Octyl-D 7.920.6 7.39
8ppm Octyl-D 1X Filtered 7.33
20ppm Octyl-D 20.10.5 2.24
20ppm Octyl-D 1X Filtered 19.64
Add detergent of known concentrations to
spin filter
Minimal filter interference
Fit for Purpose
Objective 3 met
Detergent Viral Inactivation | 20.09.201827
What happens in the final purification process?
Is the detergent cleared?
Detergent Viral Inactivation | 20.09.201828
Objective 4 – Removal of detergent in purification process
Test sample from Fc-Fusion clarified harvest – Spike in
Remove protein
ODG added
min2 4 6 8 10 12 14 16
mV
10
20
30
40
50
60
70 Post-PA + 25ppm spike inPost-PA + 10ppm spike inPost-PA
Detergent
Inactivation
Post-PA
Post-PA + 10ppm
Post-PA + 25 ppm
Sample Name Average Concentration
Sample 1 + 25ppm ODG 24.49
Sample 1 + 10ppm ODG 9.77
Sample 1 No Peaks Detected
Sample 2 + 25ppm ODG 25.2
Sample 2 + 10ppm ODG 9.8
Sample 2 No Peaks Detected
20ppm Octyl-D 20.57
20ppm Octyl-D 1X Filtered 20.03
All protein-containing samples were 1X filtered
Spike-in samples were filtered after the detergent was added to samples
Objective 4 met
Detergent Viral Inactivation | 20.09.201829
Results from Analytical Quantitation Assay of ODG
Quantitation Assay
• ODG quantitation assay developed on C18 RP-HPLC column using ELSD detector
• 8-100 ppm
Protein Removal
• Spin concentrator tested on ODG
• Removes proteins
• Does not impact detergent quantitation
Process Removal
• Detergent level is <8 ppm after PA step
1
2
3
30
Conclusions ➢ After evaluating several detergents for viral inactivation in a protein
purification cascade, ODG met all the required criteria:
▪ Viral inactivation at 6°C and 22°C
▪ < 2hour incubation
▪ Minimal impact on protein quality
➢ ODG quantitation assay developed with the following criteria:
▪ 8-100ppm quantitation range
▪ Quick and simple protein removal
▪ <8ppm of remaining detergent after protein A step
Detergent Viral Inactivation | 20.09.2018
31 Detergent Viral Inactivation | 20.09.2018
Acknowledgments
Late Stage Group
➢ Oz Ilangovan
➢ Wonki Lee
➢ Sam Guo
➢ Steven Degon
Early Stage Group
➢ Alec Murillo
➢ Vera Sellers
➢ Selena Li
➢ Ilgu Kang
Leadership
➢ Angela Lim
➢ Gene Lee
QC Group
➢ Sameth Yin
➢ Erin Williams
➢ Mike Lavallee
Analytical Group
➢ Brittany Beacham
➢ Isha Thombre
➢ Nadine Barron
➢ Dan Haq
➢ Jessica Dawson
➢ Mary Priest
➢ Michael Doty
➢ Trish Greenhalgh
➢ Corinne Miller
➢ Ushma Mehta
➢ Venkata Raman
➢ Almut Rapp
➢ Anja Licht
➢ Lisa Friedrich
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