Detergent viral inactivation and analytical quantitation of residual … · 2020. 9. 12. · 2 Detergent Viral Inactivation | 20.09.2018 Overview Detergent Viral Inactivation Detergent

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


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


• 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


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


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


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


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


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


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


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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 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


Next Step…

Can we quantitate and confirm detergent removal?

Detergent

Quantitation

Confirm

detergent

clearance


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


▪ 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


▪ 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/


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


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


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?


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


25ppm Octyl-D 25.851 3.79


10ppm Octyl-D 9.810.5 5.37


8ppm Octyl-D 7.920.6 7.39


20ppm Octyl-D 20.10.5 2.24


Add detergent of known concentrations to

spin filter

Minimal filter interference

Fit for Purpose

Objective 3 met


What happens in the final purification process?

Is the detergent cleared?


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 No Peaks Detected



Sample 2 No Peaks Detected

20ppm Octyl-D 20.57


All protein-containing samples were 1X filtered

Spike-in samples were filtered after the detergent was added to samples

Objective 4 met


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

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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



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

Documents

Detergent viral inactivation and analytical quantitation of residual … · 2020. 9. 12. · 2 Detergent Viral Inactivation | 20.09.2018 Overview Detergent Viral Inactivation Detergent