This study investigates the benefits of adding excipients during downstream processing on protein stability, chromatographic performance and viral inactivation.

1. Introduction and Objectives
The production of antibodies and Fc-fusion proteins involves several downstream processing unit
operations. The widely used purification template with Protein A chromatography, virus inactivation
at low pH, and subsequent ion exchange chromatography steps is mostly able to remove impurities
like aggregates, host-cell proteins, and viruses, which could affect the safety and efficacy of the
product.
The low pH elution during Protein A chromatography, as well as during virus inactivation may ­
induce
aggregation. Preventing protein aggregation during these unit operations instead of removing the
multimeric forms during subsequent polishing steps would be a more efficient strategy. Excipients
have shown that they can minimize aggregation levels in the final product formulation. For this ­
reason,
we have investigated the benefits of adding excipients during downstream ­
processing on
protein stability, chromatographic performance and viral inactivation.
pH
Standard
mAb
Process
Template
CEX
Protein A AEX
Figure 1:
Outline of steps involved in a general monoclonal antibody (mAb) manufacturing process.
Protein A Chromatography
HCP Distribution of mAbB over
pH-Gradient on ProSep®
Ultra Plus
HCP Distribution of mAbB over
pH-Gradient on ProSep®
Ultra Plus
Figure 2:
Influence of excipients on HCP removal during gradient elution of Protein A chromatography using ProSep®
Ultra Plus; ­
Purity of elution pool in aspect of 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.
Addition of 5% PEG4000 lead to sharper peak and provides benefit not only in the reduction
of pooling volumes but also HCP content of elution pool.
• Potential excipients (Sorbitol, Mannitol, Sucrose, Trehalose and PEG4000) have been selected by
results of low pH (pH 2.8) screening assay (Fig. 6) to be investigated further in real life DSP condi-
tions.
• Selected excipients were added to the buffer system during chromatographic run and their effect
on chromatographic performance have been observed.
• 5% PEG4000 causes a shift of sharper elution peak to lower pH during pH gradient elution, while
elution without excipient or with the use of disaccharides and polyols show broader elution peak.
• Addition of PEG4000 led also to a comparable or lower elution pool volume (Results not shown in
this poster).
• Elution behavior of the HCP’s in the presence of PEG4000 differs significantly from control and other
selected excipient conditions (Fig. 2, left).
• Purer elution pool profile with lowest HCP concentration down to 0.3 μg HCP/mg mAbB was achieved
during chromatography run with addition of 5% PEG4000 (Fig. 2, right).
• Addition of all selected excipients have no negative effect on chromatographic performance.
Virus Inactivation and Viral Clearance Study
Monomer content of mAbA pool during low pH hold
@ pH 2.80 over 60 min
Monomer content of mAbA pool during low pH hold
@ pH 2.80 over 60 min
SEC of mAbA elution pool from Eshmuno®
A at t = 60 min
Figure 3:
Results overview of low pH viral inactivation; Size exclusion analysis of elution pool of different excipient conditions (left),
mAb purity (monomer in %) of elution pool of mAb A (middle) and mAb B (right) after 60 min low pH virus inactivation
step at pH 2.8.
Addition of all selected excipients led to an increased monomer content.
0
1
2
3
4
5
6
0 10 20 30 40 50 60 70
Log
Reduction
Factors/Log
10
Duration of low pH Treatment/min
Viral Reduction Factor of
different conditions during low pH treatment
0
1
2
3
4
5
6
w/o
Excipient
0.5 M
Sorbitol
0.5 M
Mannitol
0.5 M
Sucrose
0.5 M
Trehalose
5%
PEG4000
Viral
Reduction
Factors/Log
10
Viral Reduction Factor of
different conditions after 60 min low pH treatment
w/o Excipient 0.5 M Sucrose
0.5 M Sorbitol 0.5 M Trehalose 5% PEG4000
0.5 M Mannitol
4.53
4.8
4.35 4.29
4.89
5.15
Figure 4:
Overview of viral reduction factor of different conditions during low pH treatment in viral clearance study (left) and
­
summarized results of 60 min low pH treatment (right).
*
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
Addition of selected excipients have no negative effect on viral inactivation. Slight improve-
ment by addition of Sorbitol, Trehalose and PEG4000.
• Decreased aggregate content (dimers) could be observed in all excipients conditions (Fig. 3, left).
• Addition of all selected excipients led to an increased monomer content up to 6.3% (Fig. 3, middle
and right). The results are well in line with excipient screening results.
• Based on EMA guideline in concideration of ICH Q5A guideline, Viral reduction of all tested ­
conditions
have been classified as highly effective (viral reduction factor 4log10
, Fig. 4).
• Slight improvement by addition of Sorbitol, Trehalose and PEG4000 could be observed in viral clear-
ance study (Fig. 4).
• Addition of selected excipients have no negative effect on viral reduction.
Dynamic Binding Capacity of CEX
Dynamic Binding Capacity (DBC) of CEX column (Eshmuno®
CPX, Merck)
at 10% breakthrough under different excipient conditions.
w/o Excipients
500 mM Mannitol
500 mM Sucrose
500 mM Trehalose
5% PEG4000
500 mM Sorbitol
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-Signal
percentage
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, Merck)
at 10% breakthrough under different excipient conditions.
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-Signal
percentage
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, Merck)
at 10% breakthrough under different excipient conditions.
Figure 5:
Dynamic Binding Capacity (DBC) of CEX column (Eshmuno®
CPX, MilliporeSigma) at 10% breakthrough under different
additive conditions.
The addition of excipients don‘t affect the dynamic binding capacity in cation exchange
­
chromatography. Slight improvement of DBC with addition of 500 mM Sorbitol.
• The capacity was unaffected with addition of PEG4000, Trehalose and Mannitol (DBC 10% ca. 140–
145 mg/mL CV).
• Slight reduction on binding capacity was observed in additive condition of 500 mM Sucrose.
• Nevertheless, the presence of all additives don‘t negatively affect the process condition, because
safety factor is generally used on CEX process (e.g. 80% of the 10% breakthrough).
Screening Assay
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 500 mM ionic excipient
Control_50 mM NaCl 50 mM NaCl_500 mM ionic excipient
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 500 mM
Sorbitol
500 mM Ionic
excipient
w/o Excipient 500 mM
Sorbitol
500 mM Ionic
excipient
acidic buffer pH 2.8 acidic buffer pH 2.8+
50 mM NaCl
Tm-Values/°C
Figure 6:
Exemplary results of screening in kinetic SEC diagram (left) and melting temperature (Tm) diagram by nanoDSF
(right) showing stabilizing effect of certain excipients (Sorbitol and an ionic excipient as negative control) on mAbA during
low pH treatment.
Addition of suitable excipients during aggregation kinetic assay prevents ­
formation of
aggregats stabilization of mAbA during low pH stress.
• To determine the influence of excipients in Protein A chromatography and virus inactivation, a low
pH screening assay was established.
• Potential Excipients (Sorbitol, Mannitol, Sucrose, Trehalose and PEG4000) during screening
in acidic conditions were investigated further in real life condition in Protein A chromatography and
viral clearance study.
• Addition of suitable excipient (e.g. Sorbitol) prevents formation of aggregates (Fig. 6, left) and
­
increase Tm up to 1.5 °C (Fig. 6, right) which indicates stabilization of mAbA during low pH stress
condition.
• Polyols (e.g Mannitol, Sorbitol, Innositol) and Sugars (e.g. Sucrose and Trehalose) and PEG4000
were most effective excipients in low pH screening condition with and without the absence of
­sodium chloride.
Improving Protein Purification: ­
Application of Excipients
in ­
Downstream Processing
Supriyadi Hafiz1
, Tanja Henzler1
, Christoph Korpus1
, Romas Skudas1
, Bahar Cebi1
, Carolin Stange2
, Christian Frech2
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
MilliporeSigma, the Vibrant M, Eshmuno, 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.
Lit. No. MS_PS7669EN 03/2021
The Life Science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Conclusion
In this study, we were able to confirm the addition of excipients can have a beneficial effect
for the purification during Protein A chromatography and virus inactivation by stabilizing
proteins, seeing that the level of monomer can be increased up to 6.3%, reduction of volume
and HCP content of elution pool, without harming the subsequent chromatographic steps.
1
Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany.
2
Institute for Biochemistry, University of Applied Sciences Mannheim, Paul-Wittsack-Straße 10, 68163 Mannheim, Germany.