Improving Downstream Processing: Application of Excipients in DSP

The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Improving
Downstream
Processing:
Application of
Excipients in DSP
Webinar
Prof. Dr. Christian Frech
November 14th , 2019

The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

Agenda
Introduction and Objectives
Excipient Screening
Excipients for Protein A chromatography
Viral Inactivation Study4
3
2
1
Excipients for CEX chromatography5
6 Summary
3 Excipients for Protein Stabilization in DSP - Webinar | 14.11.2019

Excipients for Protein Stabilization in DSP - Webinar | 14.11.2019
Introduction and Objectives
 During Downstream Processing (DSP) of monoclonal antibodies (mAbs), pH shifts and/or changing
conductivities often lead to aggregation, fragmentation or other chemical modifications
 Application of high quality excipients could lead to improved protein stability as well as solubility
during DSP
Investigation of excipients on product stability and chromatographic performance during
downstream processing
pH
5
StandardmAbProcess
Template
CEXProtein A AEX

Approach:
Evaluating excipient effects on the stability of acid-exposed monoclonal antibodies by simulating protein A elution and
virus inactivation condition in DSP → Screening of excipients
Screening Conditions:
• Model Proteins: 2 IgG1 mAbs (mAbA and mAbB)
• Excipients: Sugars, polyols, amino acids, surfactants and polymers
• Condition 1 (condition without NaCl): 100 mM acidic buffer pH 2.8 and +/- excipients
• Condition 2 (condition with addition of NaCl): 100 mM acidic buffer pH 2.8; 50 mM NaCl and +/- excipients
• pH Aggregation Shift Assay: The pH of mAb solution was adjusted by dilution to pH 2.8 +/- NaCl and incubated at RT for 2h
• Screening Readout: Δ Difference of monomer content and Tm-values between samples (with excipient) and control (without excipient) at t = 2 h
Excipient Screening set-up
AnalyticsAcidic
pH Shift
IgG1 mAbs in storage buffer mAb sample at pH 2.8
+/- 50 mM NaCl and +/- excipients
Kinetic SE-HPLC & nanoDSF
Source: www.agilent.com; www.nanotempertech.com

Unstressed mAbB in storage buffer
Control; without excipient
with addition of 0.5 M sorbitol
with addition of 0.5 M ionic excipient
with addition of 0.5 M sucrose
Exemplary SEC
chromatograms of
mAbA +/- excipients
after 2h incubation
acidic buffer pH 2.8
Addition of suitable excipients (e.g. sucrose and sorbitol) leads to a decreased
aggregate content (dimers/multimers) during low pH stress.
SEC Analysis of Aggregation Kinetics
HMW
Monomer
HMW

Addition of suitable excipient (e.g. Sorbitol) during aggregation kinetic assay prevents
formation of aggregats → stabilization of mAb during low pH stress.
Excipient Effects on Aggregation Kinetics
85
90
95
100
0 20 40 60 80 100 120 140
Monomer/%
Time /min
Control Control_50mM NaCl
500mM Sorbitol 50mM NaCl_500mM Sorbitol
Overview of kinetic SEC results of Monomer of mAbA
With addition of Sorbitol at pH 2.8
Overview of kinetic SEC results of Monomer of mAbA
With addition of an ionic excipient at pH 2.8
70
80
90
100
0 20 40 60 80 100 120 140
Monomer/%
Time /min
Control 500mM ionic excipient
Control_50mM NaCl 50mM NaCl_500mM ionic excipient

• Tm Analysis by Nano Differential Scanning Fluorimetry (nanoDSF) monitors protein unfolding
by changes in its intrinsic fluorescence at 330 and 350nm wavelength
• Application of thermal ramps between 15 – 95 °C for 48 samples in parallel
• Sample requirements: 10 µl sample, 5 µg/ml to 250 mg/ml protein concentration
Determination of Conformational Stability by nanoDSF
Determination of Melting Temperature (Tm) of mAbs
Schematic representation of nanoDSF workflownanoDSF Technology –Thermal Unfolding
Source: www.nanotempertech.com

Addition of suitable excipient (e.g. Sorbitol) increase the Tm up to 1.5 °C, which indicates stabilization
of mAbA during stress condition.
NanoDSF results correspond also to SEC aggregation kinetic results
Excipient Effects on Conformational Stability
Overview of nanoDSF results of mAbA
With addition of Sorbitol and an ionic excipient at pH 2.8
53,0
54,5
50,6
52,2
53,8
49,8
40
45
50
55
60
w/o Excipient 500mM
Sorbitol
500mM Ionic
excipient
w/o Excipient 500mM
Sorbitol
500mM Ionic
excipient
acidic buffer pH 2.8 acidic buffer pH 2.8+
50mM NaCl
Tm-Values/°C

Aggregation Kinetic Assay and NanoDSF
Excipient Screening Results: Overview
Sample Analytics
Readout
Polyol Sugars Amino Acids Surfactans & Others
Sorbitol Mannitol Sucrose Trehalose Ionic
excipient
Arginine
HCl
Lysine
HCl
Alanine Glycine Glutamate PS20 PS80 PEG
4000
Poloxmer
188
mAb A
Acidic buffer
pH 2.8
without NaCl
Condition 1
Δmonomer
(Kinetic SEC)
ΔTm
(nanoDSF)
Acidic buffer
pH 2.8+
50 mM NaCl
Condition 2
Δmonomer
(Kinetic SEC)
ΔTm
(nanoDSF)
mAb B
Acidic buffer
pH 2.8
without NaCl
Condition 1
Δmonomer
(Kinetic SEC)
ΔTm
(nanoDSF)
Acidic buffer
pH 2.8+
50 mM NaCl
Condition 2
Δmonomer
(Kinetic SEC)
ΔTm
(nanoDSF)
Stabilization effect
of excipient is significant
-Δmonomer ≥ 0.5% and
-ΔTm ≥ 0.5°C
No effect or slight negative effect of excipient
on protein stability is observed
-5%≤ ∆Monomer < 0.5%
-5°C ≤ ∆Tm ≤ 0°C
Strong negative effect of excipient
on protein stability is observed
-5%≤ ∆Monomer
-5°C ≤ ∆Tm
Potential Excipients (Sorbitol, Mannitol, Sucrose, Trehalose and PEG 4000) during
screening in acidic conditions were investigated in Protein A chromatography.

Excipients for
Protein A
Chromatography

Protein A Chromatography
14
StandardmAbProcess
Template
CEXProtein A AEX

• Model Proteins: 2 IgG1 mAbs (clarified Harvest of mAbA 0.94 g/l and mAbB 1.45 g/l)
• 3 different Protein A resins
• Based on their potential on stability results of in vitro screening assay, Sucrose, Trehalose, Sorbitol, Mannitol and PEG4000
(500 mM or 5% for PEG4000) were selected for further investigation on chromatographic performance and product stability using
Protein A chromatography
• Selected excipients were added to the buffer system during chromatographic run
Protein A Chromatography Conditions
Selected Protein A Columns Selected Excipients
Eshmuno® A; 0.8x2 cm; 1 ml 500 mM Mannitol
ProSep® Ultra Plus; 0.8x2 cm; 1 ml 500 mM Sorbitol
Agarose-based Protein A Resin; 0.8x2 cm; 1 ml 500 mM Sucrose
500 mM Trehalose
5% PEG4000
Experiment Parameters:
Eshmuno® A Resins ProSep® Ultra Plus Resins

Chromatographic Run of IgG1 mAbs on Protein A Columns
Selected excipients were added to the buffer system during chromatographic run
and their effect on chromatographic performance has been observed
System: ÄKTApurifierTM GE Healthcare
Column: Eshmuno® A (0.8x2 cm), 1 mL
Flow: 120 cm/h (1 mL/min with Eshmuno® A)
Buffer system: A: 100 mM Citrate, pH 5.5; B: 100 mM
Citrate, pH 2.75 (excipients added as indicated in buffer A and
B);
Gradient: 30 CV; The column was equilibrated with 100 mM
Citrate pH 7.0 (Wash pH 7.0 3 CV after injection, then step to
buffer A pH 5.5)
Sample: mAbA, clarified harvest, 0.943 mg/mL
Sample load: 30 mg/mL CV
Chromatographic run of mAbA on Eshmuno® A
Equilibration, Inject
and 1. Wash without
excipient @ pH 7.00
Introduction of excipients
during 2. Wash and Elution
– pH gradient 5.50 – 2.75

Size exclusion analysis of the collected fractions
Chromatographic run of mAbA on Eshmuno® A
The collected fractions during elution show a high purity – salts and other
impurities have been separated in the flowthrough
+/- excipient

Improvement of mAbA Elution Performance by Excipients
Eshmuno® A Performance on
mAbA ± Excipient
ProSep® Ultra Plus Performance on
mAbA ± Excipient
Agarose-based Resin Performance on
mAbA ± Excipient
Addition of 5% PEG4000 causes a shift of sharper elution peak to lower pH, while
elution without the use of excipient or with the use of disaccharides and polyols show
broader elution peak.

Improvement of Chromatography Performance on mAbA
Addition of PEG4000 led to a comparable or lower elution pool volume
Collected Elution Pool Volume of mAbA
during Protein A chromatography
Peak Area of Purified mAbA
• Addition of all selected excipients have no negative effect on chromatographic performance
• mAb elution was pooled according to UV280 > 30 mAU

Improvement of mAbB Elution Performance by Excipients
Eshmuno® A Performance on
mAbB ± Excipient
ProSep® Ultra Plus Performance on
mAbB ± Excipient
Agarose-based Resin Performance on
mAbB ± Excipient
Addition of 5% PEG4000 causes a shift of sharper elution peak to lower pH, while
elution without the use of excipient or with the use of disaccharides and polyols show
broader elution peak.

Improvement of Chromatography Performance on mAbB
Addition of PEG4000 led to a comparable or lower elution pool volume
Collected Elution Pool Volume of mAbB
Peak Area of Purified mAbB
• Addition of all selected excipients have no negative effect on chromatographic performance
• mAb elution was pooled according to UV280 > 30 mAU

Improvement of elution performance with increasing PEG
concentrations
Increasing the PEG4000 concentration leads to a sharper peak profile
Chromatographic run of mAbA on Eshmuno® A Chromatographic run of mAbB on Eshmuno® A

23
Flowthrough
Wash pH 7
Wash pH 5.5
Gradient
Original mAbB sample
Influence of Excipients on HCP removal
System: ÄKTApurifierTM GE Healthcare
Column: Eshmuno® A (0.8x2 cm), 1 mL
Flow: 120 cm/h (1 mL/min with Eshmuno A)
Buffer system: A: 100 mM Citrate, pH 5.5; B: 100 mM Citrate, pH
2.75 (excipients added as indicated in buffer A and B);
Gradient: 30 CV; The column was equilibrated with 100 mM Citrate
pH 7.0 (Wash pH 7.0 3 CV after injection, then step to buffer A pH 5.5)
Sample: mAbB, 1.45 mg/mL, clarified harvest
Sample load: 30 mg/mL CV
Chromatographic run of mAbB on Eshmuno® A

HCP Distribution of mAbB over pH - Gradient on
Eshmuno® A
More HCPs were eluted in the rear part of the gradient in a large peak, which means HCP elution
was shifted to slightly lower pH condition, especially for PEG4000

HCP Distribution of mAbB over pH-
Gradient on Eshmuno® A
Gradient on ProSep® Ultra Plus
Gradient on Agarose-based Resin
• Elution behavior of the HCP's in the presence of PEG4000 differs significantly from control and other selected
excipient conditions.
• More HCPs were eluted in the rear part of the gradient in a large peak, which means HCP elution was shifted in
slightly lower pH condition.
• Purity of elution pool with respect to HCP content was analyzed by comparison of HCP content of elution pool
from collected fractions based on UV280 collection criterion of >30 mAU, with total mAb content during pH
gradient.

Improvement of HCP Reduction with 5% PEG4000 on mAbB
Gradient on Eshmuno® A
Gradient on ProSep® Ultra Plus
Gradient on Agarose-based Resin
• Purity of elution pool was analyzed by comparison of HCP content of elution pool from collected fractions based on UV280
collection criterion of >30 mAU, with mAb content of elution pool
Purer elution pool profile with lowest HCP concentration down to 0.23 µg HCP/mg mAbB was
achieved during chromatography run with addition of 5% PEG4000.

 Virus inactivation step was performed after Protein A chromatography by lowering the pH of the elution
pool to pH 2.8
 Aggregation kinetics were monitored by analytical SEC
Viral inactivation
pH
28
StandardmAbProcess
Template
CEXProtein A AEX
Investigation of excipients for stabilization of the monomer during low pH hold
and their effect on viral inactivation rates

Size exclusion analysis of elution pool during virus inactivation
SEC of mAbA elution pool from Eshmuno® A at t = 0 min SEC of mAbA elution pool from Eshmuno® A at t = 60 min
Addition of all selected excipients led to an increased monomer content

Improvement of mAb Stability
Monomer content of mAbA pool during low pH hold
@ pH 2.80 over 60 min
Monomer content of mAbB pool during low pH hold
@ pH 2.80 over 60 min
 Even during the pH adjustment to pH 2.80 the addition of excipients leads to a stabilization of monomer
content
Addition of all selected excipients led to an increased monomer content up to 6.3%.
The results are well in line with excipient screening results

Viral Inactivation Assessment
Experiment Parameters:
• Model virus: Xenotropic Murine leukaemia virus (MLV)
• Model protein: mAbB (130 mg/ml) → diluted to 10 mg/ml with selected formulation during low pH treatment
• 5 Excipients were tested for their influence in viral inactivation step and compared with the basic experiment without addition of excipient
• Low pH treatment buffer: acidic buffer pH 3.6 with and without selected excipients
• Duration of low pH treatment: 0, 5, 15, 30 and 60 minutes
• Viral load: ca. 6 log10 TCID50/ml
• Objective: to confirm that selected excipients do not negatively affect the virus inactivation
31
No. Excipients
1 500 mM Sorbitol
2 500 mM Mannitol
3 500 mM Sucrose
4 500 mM Trehalose
5 5% PEG4000

Excipients don’t negatively affect viral inactivation
0
1
2
3
4
5
6
0 10 20 30 40 50 60 70
ΔLogReductionFactors
/Log10
Duration of low pH Treatment /min
Viral Reduction Factor of
different conditions during low pH treatment
w/o Excipient 0.5M Sorbitol
0.5M Mannitol 0.5M Sucrose
0.5M Trehalose 5% PEG4000
32
*Examples of effective virus reduction combined with reduction factor based on EMA
guideline in consideration of ICH Q5A guideline
Reduction Factor Effectiveness*
≤ 1 log10 Not significant
1-2 log10 Indicative/contributable
2-4 log10 Moderate
> 4 log10 High
4,53
4,8
4,35 4,29
4,89
5,15
0
1
2
3
4
5
6
w/o
Excipient
0.5M
Sorbitol
0.5M
Mannitol
0.5M
Sucrose
0.5M
Trehalose
5%
PEG4000
ViralReductionFactors
/Log10
Viral Reduction Factor of
different conditions after 60 min low pH treatment
Slight improvement by addition of Sorbitol,
Trehalose and PEG4000.

Excipients for
CEX
Chromatography

Objective
 Subsequent to low pH virus inactivation the pH is adjusted to match loading conditions for cation
exchange chromatography step
 Application of high quality protein excipients could influence the dynamic binding capacity of
cation exchange chromatography
pH
34
StandardmAbProcess
Template
CEXProtein A AEX

Dynamic Binding Capacity of CEX Column in the Presence of Excipients
35
The addition of excipients don‘t affect the dynamic binding capacity in cation exchange
chromatography
Column: Eshmuno® CPX (0.5x5 cm), 1 mL
System: Äkta Purifier
Flow: 92 cm/h (0.3 mL/min) at elution; 61 cm/h (0.2 mL/min) at binding
Buffer system: A: 10 mM Sodium Citrate, pH 5.0; B: 10 mM Sodium Citrate,
pH 5.0 + 1 M NaCl (excipients added as indicated in buffer A and B);
Gradient: 20 CV;
Sample: mAbB, ~5.25 mg/mL,
Post Prot A, diluted in buffer A with or without Excipients
(concentrations of excipients in diluted samples: 4.7% PEG4000 or 465 mM
sugar excipients)0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
UV-Signalpercentage
Protein load /mg
w/o Excipient 5% PEG4000 500 mM Sucrose
500 mM Mannitol 500 mM Sorbitol 500 mM Trehalose
Dynamic Binding Capacity (DBC) of CEX column (Eshmuno®
CPX) at 10% breakthrough under different excipient conditions.

Dynamic Binding Capacity of CEX Column in the Presence of Excipients
36
• Slight reduction on binding capacity was
observed in excipient condition of 500 mM
Sucrose
• The capacity was unaffected with addition of
PEG4000, Trehalose and Mannitol (DBC 10%
ca. 140-145 mg/ml CV)
Slight improvement of DBC with addition of 500mM Sorbitol
Dynamic Binding Capacity (DBC) of CEX column (Eshmuno® CPX) at
10% breakthrough under different excipient conditions.

Effect of Excipients on separation in CEX
Several publications have described the effect of PEG and the superior
antibody/aggregate separation in cation exchange chromatography.

Summary
• Excipient screening: selected excipients, 500 mM Mannitol, 500 mM Sorbitol,
500 mM Sucrose, 500 mM Trehalose and 5% PEG4000 were effective to stabilize
mAbs during simulated low pH virus inactivation step.
• Protein A screening:
o Addition of 5% PEG4000 lead to sharper peak
o Furthermore 5% PEG4000 provides a significant benefit not only in the reduction of
pooling volumes but also HCP content of elution pool.

Summary
• Virus inactivation study:
o Up to 6.3% Monomer increase has been achieved due to addition of excipients
during virus inactivation (60 min incubation at pH 2.8 after Protein A chromatography)
o Addition of selected excipients have no negative effect on viral reduction factor
➢ Slight improvement by addition of Sorbitol, Trehalose and PEG4000
• Cation exchange chromatography:
o Addition of selected excipients have no negative effect on DBC in CEX Column
➢ Slight improvement by addition of Sorbitol
Emprove® excipients can be used to stabilize proteins during low-pH stress
e.g. Protein A chromatography and virus inactivation.

christian.frech@stw.de
Prof. Dr. CHRISTIAN FRECH
The vibrant M, Eshmuno, Emprove, ProSep and SAFC are trademarks of Merck KGaA,
Darmstadt, Germany or its affiliates. All other trademarks are the property of their
respective owners. Detailed information on trademarks is available via publicly
accessible resources.
© 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
tanja.henzler@emdgroup.com
Dr. Tanja Henzler

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