Application Note
USTR 2795
Simple, Efficient 96-Well Plate High Throughput
Screening Lab Method to Optimize the Use of
Mixed-Mode Chromatography Sorbents for the
Purification of a Monoclonal Antibody
Summary
Introduction
The benefits of the unique selectivity and
chemical stability offered by mixed-mode
chromatography for the purification of
monoclonal antibodies (MAbs) are well
established. Rapid determination of
optimum operating conditions earlier
in process development maximizes the
value of these industrial adsorbents.
Current platforms for MAb purification,
mostly relying on Protein A as a first
capture step, consist of two to three
chromatography steps. These platforms
are robust and efficient but significantly
increase downstream purification costs.
To decrease manufacturing costs,
industry is increasingly considering the
use of alternative purification schemes
that could avoid the use of affinity on
Protein A sorbents, in addition to reducing
the number of steps.
This study describes the use of low cost,
easy-to-use bench technology to perform
high throughput screening (HTS) to optimize
separation conditions for the purification
of monoclonal antibody using Pall mixedmode chromatography sorbents.
Screening conditions and analytical results
were completed in three days. Subsequent
transfer of conditions to a dynamic column
system (1 mL column) were completed
in two days. The complete optimization
process can therefore take place in a
single week.
The study shows that the optimized use of
HEA HyperCel™ mixed-mode sorbent after
a first capture on Protein A could lead to a
robust two-step MAb purification process.
Mixed-mode chromatography sorbents
could fix these issues. Pall MEP, HEA, and
PPA HyperCel mixed-mode sorbents carry
mixed-mode synthetic ligands immobilized
on a robust and scalable matrix (HyperCel)
that confers high porosity, chemical stability
and excellent stability to harsh cleaning-inplace conditions (1 M NaOH). The ligands
include aliphatic (HEA – hexylamine) and
aromatic (MEP – mercaptoethylpyridine,
PPA – phenylpropylamine) groups (Figure 1).
Figure 1
Ligand structures and schematic representation of the
adsorption (mostly relying on hydrophobic interactions)
and desorption (mainly driven by electrostatic repulsion)
mechanism of HyperCel mixed-mode sorbents.
These mixed-mode ligands contain hydrophobic (aliphatic
chain or aromatic ring) and electrostatic (amine group,
hydroxyl hydrogen bonding groups) components. For HEA
and PPA HyperCel ligands, electrostatic interactions play a
role in protein adsorption at low to moderate conductivity,
whereas protein capture by hydrophobic interaction is
favored by adding salt to the sample. Protein elution is
also controlled by both types of interaction.
This unique mechanism of separation on mixed-mode
chromatography sorbents offers new selectivities that
can reduce the number of purification steps for MAb
purification processes. However, to achieve the best
separation results, this type of separation requires a
good optimization of pH and conductivity conditions.
In order to provide a simple tool to optimize the use
of mixed-mode sorbents in a MAb purification process,
a high throughput screening method based on DoE
combined with 96-well plate experiments was developed
(Figure 2).
A mixed-mode sorbent, HEA HyperCel, was used as a
second step of a MAb purification after capture on Protein A.
Optimum pH and conductivity were screened to obtain the
best performances (yield of recovery and HCPs removal).
2
Figure 2
Optimization of chromatographic run conditions using the
combination of DoE and experiments on microplates.
Materials and Methods
Analytical Methods
Quantification of HCP Host Cell Proteins (HCPs)
Quantification of HCPs was done by ELISA using a kit
from Cygnus Technologies (HCP Host Cell Proteins, #F015).
Samples were diluted in a range of 1/1 to 1/10000
depending on the step analyzed.
Protein Quantification
Antibodies were quantified throughout the purification
steps either by evaluation of the peak area obtained during
SEC-HPLC analysis or by A280nm measurement using a
Nanodrop* 1000 (Thermo Scientific). Protein concentrations
were calculated using a mass extinction coefficient of
13.7 at 280 nm for a 1% (10 mg/mL) IgG solution.
Preparation of a Post-Protein A Pool
As HEA HyperCel sorbent was used as a second
purification step, a post-Protein A pool was prepared
using a Protein A agarose sorbent. 120 mL of HCP
clarified cell culture supernatant containing 1.7 g/L MAb
(210 mg total MAb) were loaded onto the Protein A
column at 28 mg/mL of sorbent and the MAb was
eluted using 10 mM glycine-HCl, pH 3.5. A total of
180 mg post-Protein A MAb pool was recovered
(85% yield) and used to screen the best conditions
to use on HEA HyperCel sorbent.
The HCP content was 400 ppm, whereas the HCP
concentration in the initial MAb feedstock was 60,000 ppm.
Design of Experiments (DoE) for Screening Conditions on HEA HyperCel Sorbent in 96-well Plates
Using statistical models, DoE allows the determination of
the effects of different factors on the analyzed responses,
and the best run conditions. The factors studied were
pH and conductivity for load, wash and elution. The
responses analyzed were MAb yield of recovery and
CHOPs removal. Considering the total number of factors
to be studied, we performed a first screening by varying
the load and wash conditions. We used a 4 factors
D-Optimal response surface design generated using
the Modde◆ software to establish the list of experiments
to be performed.
The design chosen generated a series of 27 experiments,
including one experimental condition performed in
triplicate to assess the experimental reproducibility. The
experimental points with a wash pH higher than the load
pH were removed from the list, as raising the pH would
result in increasing interactions to the mixed-mode resin,
which is not wished during a washing step. As several
points were excluded, the design was slightly modified in
order to fully explore the design region contained within
the factor testing ranges. The final list of experimental
points is shown in Table 1a.
A second set of experiments, listed in Table 1b, was then
defined to optimize elution pH conditions. Three different
elution pH were used.
The responses were subsequently processed using the DoE
software to generate a valid statistical model, and optimum
conditions to be used on mixed-mode sorbents were identified.
Table 1a
DoE optimization of conditions of use of HEA HyperCel
sorbent as second step post-protein A.
Results of
96-well Plate
Screening Conditions
Experiments
Elution
Load
Wash
yield
Run cond
Load cond
Wash (% of HCP
Exp # order (mS/cm) pH
(mS/cm) pH
bound) (ppm)
HEA1 8
8
6.5 15.0
6.5
75.8
2.3
HEA2 14
15
6.5 2.5
6.5
18.2
ND
HEA3 20
15
6.5 15.0
6.5
74.4
4.1
HEA4 19
15
7.0 2.5
7.0
65.5
ND
HEA5 21
15
7.0 15.0
6.5
76.4
3.1
HEA6* 25
15
7.0 15.0
7.0
80.5
2.5
HEA7* 26
15
7.0 15.0
7.0
76.4
2.5
HEA8* 27
15
7.0 15.0
7.0
80.2
2.9
HEA9 2
8
7.5 2.5
6.5
31.8
ND
HEA10 4
8
7.5 15.0
7.0
66.4
ND
HEA11 5
8
7.5 2.5
7.5
78.8
2.6
HEA12 6
8
7.5 2.5
7.0
66.2
ND
HEA13 9
8
7.5 15.0
6.5
70.7
2.0
HEA14 11
8
7.5 15.0
7.5
96.9
2.0
HEA15 15
15
7.5 2.5
6.5
40.8
ND
HEA16 17
15
7.5 2.5
7.5
69.8
ND
HEA17 23
15
7.5 15.0
7.5
53.2
ND
Table 1b
Conditions
Exp #
HEA18
HEA19
HEA20
Elution pH
5.0
5.5
6.0
Results of 96-Well Plate Experiments
Elution yield
(% of bound)
HCP (ppm)
81.9
11.9
68.2
11.5
30.1
30.1
a. Optimization of pH and conductivity conditions of load and wash
steps. Results obtained regarding elution yield and HCP content
are presented in the right part of the table. The HCP content was
determined only for the experiments showing an elution yield > 75%.
Elution conditions used for all experiments were 50 mM sodium
acetate pH 5.0
b. Optimization of elution conditions. The indicated elution pHs were
tested for the elution of HEA HyperCel sorbent while applying the
optimal conditions previously determined for the load and wash steps.
*Triplicate point to assess experimental reproductibility
ND = Not Determined
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3
High Throughput Screening on 96-Well Plate
Screening of multiple conditions for the optimization of
purification step on mixed-mode sorbents was performed
using an AcroPrep™ 96-well filter plate. The selected
sorbent was equilibrated and re-suspended as a 50%
slurry in the equilibration buffer and the desired amount
of slurry was dispensed into the wells to a final volume
of 50 µL per well. The liquid was then aspirated using a
multi-well plate vacuum manifold. A sequence mimicking
a chromatographic run was then performed on the plates
following the steps indicated in Table 2. For each step of
the sequence, the corresponding solution was pipetted
into the wells. The microplate was then covered and
incubated under shaking for the indicated time. After
incubation, the liquid was drained from the wells using
the vacuum manifold and collected in a 96-well plate;
then fractions were analyzed.
Table 2
General sequence used for microplate experiments. For each
step the buffer is determined according to the experimental
design specific to the sorbent and conditions tested.
Equilibration Load
Volume (µL) 200 200
According to
experiment
design
Incubation 5
5
60
time (min)
Wash
Elution
200 200 200 200
5
5
10
10
Evaluation of Statistical Model Validity
The data gathered from the analysis of 96-well plate
collected fractions was used to generate a statistical
model using the DoE software.
The model validity was checked by analyzing the R2
and adjusted R2 values which represent the proportion
of variation in the response that is explained by the model.
A model was considered as good if both R2 and adjusted
R2 were >85%.
Transfer to Column of Optimized Conditions
The optimal conditions predicted from the statistical model
analysis were transferred to 1 mL PRC prepacked column
(Pall Life Sciences).
The column was equilibrated in 10 mM Na Phosphate,
pH 7.0 adjusted to a conductivity of 8 mS/cm by addition
of NaCl. 8 mg of the post-Protein A pool equilibrated to
pH 7.0 and to a conductivity of 8 mS/cm were loaded
onto the column. After a washing step using the same
buffer as the equilibration step, the bound proteins were
eluted in 50 mM Na Acetate, pH 5.0.
4
Results and Discussion
Optimization of Conditions to be Applied to HEA
HyperCel Sorbent
Generation of Statistical Model and Evaluation of
its Validity
The results generated during the screening of load and
wash conditions for HEA HyperCel sorbent on 96-well
plates are shown in Table1a. These results were analyzed
using response surface modelling to generate models
representing the variations of elution yield and HCP content
as a function of load and wash pH and conductivity.
Satisfying models, fulfilling the validity requirements, were
obtained from the software for both elution yield and HCP
content. No non-significant points were identified in the set
of obtained data.
Models meeting the requirements (R2 and adjusted R2 >85%)
were used to generate the contour plots (Figures 3 and 4)
illustrating the effects of the load and wash conditions on
the elution yield and HCP amount.
Optimization of Conditions to be Applied to HEA
HyperCel Sorbent
Screening for optimal conditions for HEA HyperCel sorbent
following a capture step on Protein A was performed in
varying pH and conductivity for the load, wash and elution.
In order to simplify the DoE, the optimization was split in
two parts. In a first part, four variables (load pH, load
conductivity, wash pH, wash, and conductivity) were
screened to optimize load and wash conditions only.
In a second part, the elution conditions were tested using
three different elution pH.
Optimization of Load and Wash Conditions Using DoE
The optimization of load and wash conditions were
explored using 96-well plate and the collected fractions
were analyzed for MAb yield of recovery and HCP content
(Table 1a).
For all the conditions tested, no significant amount of
antibody was detected in the flowthrough, showing that
all conditions tested were favorable to the binding of the
MAb to the sorbent.
The effects of the load and wash conditions on the elution
yield and the HCP content were analyzed. It appeared
that either both parameters could dramatically change
depending on the conditions used (Figures 3 and 4).
Analysis showed that the elution yield was better with
lower load conductivities and with an intermediate load
pH of 7.0. For wash conditions, a combination of high
wash pH and high wash conductivity allows the best
recovery of MAb in the elution. Regarding the HCP
content, the loading pH shows a bell shape influence,
with the most favorable conditions at pH of 7.0. In
addition, increasing the load conductivity leads to a higher
HCP content in the elution fraction (high salt content
apparently favors the binding of HCP on the sorbent).
The wash conductivity has been explored in a broader
range than the load conductivity, and the highest wash
conductivity values lead to the lowest HCP content. The
wash pH has a similar bell shape influence as the load pH
on the HCP content, with the most favorable condition at
pH 7.0.
Figure 3
Contour plots showing the effect of load and wash
conditions on MAb elution yield obtained using HEA
HyperCel sorbent as second step post-Protein A.
After having optimized the model to fit with the data
measured, the optimum conditions were determined so
that we could have a yield of recovery over 80% and an
HCP content below 10 ppm. The contour plots presented
in Figure 5 show the design space available for the load
and wash conditions where both criteria are met (area in
white). Based on the model used, the optimal conditions
determined for the use of HEA HyperCel sorbent postProtein A were pH 7.0, conductivity 8 mS/cm for the load
and pH 7.0, conductivity 15 mS/cm for the wash. This
model also predicted a yield of 82% and a HCP content
below 10 ppm using these conditions for load and wash.
Figure 5
Overlapping contour plots for elution yield and HCP content.
The white area shows the design space available to combine acceptable
values for the two factors. For this analysis, the acceptable values were
set as follows : HCP < 10 ppm and elution yield > 80%. The graph on the
left shows the feasible region for load pH and conductivity with defined
wash conditions (pH 7.0, conductivity 15 mS/cm) and the graph on the
right shows the feasible region for wash pH and conductivity with defined
load conditions (pH 7.0, conductivity 8 mS/cm).
Figure 4
Contour plots showing the effect of load and wash
conditions on HCP content in elution obtained using HEA
HyperCel sorbent as second step post-Protein A.
Optimization of Elution Conditions
After determination of the optimal load and wash conditions,
the optimal elution pH for HEA HyperCel sorbent A was
determined. Several elution pHs were tested using 96-well
plate (Figure 6), and the optimal elution pH was found to
be pH 5.0. At this pH, HCP content was close to 10 ppm
and yield was 82%, confirming the model predictions.
Increasing the elution pH led to increased HCP content
and decreased elution yield.
The use of DoE in combination with experiments
performed in 96-well allowed the determination, in two
steps, of optimal operating conditions to be used on
HEA HyperCel sorbent as a post-Protein A step.
These conditions could next be confirmed by using
them on columns.
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5
Figure 6
Test of elution pH for HEA HyperCel sorbent used as
second step post-protein A.
Figure 7
Chromatogram of the HEA HyperCel sorbent run on
column applying the optimal conditions determined by
DoE after capture on Protein A affinity sorbent (1 mL PRC
prepacked column with HEA HyperCel sorbent.)
The elutions were carried out using 50 mM Na acetate for pH 5.0 and
5.5, and 50 mM Na Phosphate for pH 6.0. The blue line represents the
elution yield as percentage of MAb bound on the sorbent and the green
bars represent the HCP content in ppm.
Load : 8 mg/mL of sorbent
Transfer of Optimized Conditions on 1 mL
Prepacked Column
The optimal conditions determined above were applied
to a 1 mL PRC prepacked column with HEA HyperCel
sorbent; the chromatogram is shown in Figure 7. Analysis
of the elution fractions showed a yield of 83% and a HCP
content below 10 ppm.
Results indicate a very good correlation between the
prediction from the DoE/96-well plate experiments and
the data obtained on column. This clearly shows that
high throughput screening (HTS) can simplify and
accelerate the optimization of pH and conductivity
conditions to apply on mixed-mode sorbents. This work
could lead to the development of an efficient two-step
purification process for the MAb tested.
Conclusions
Mixed-mode chromatography relies on multiple types
of protein-ligand interactions and requires optimization.
We used DoE in combination with 96-well plates to
simplify and significantly accelerate this optimization as
the complete work was performed in a single week.
This approach demonstrates the benefits of using mixedmode chromatography in a MAb purification process.
A two-step purification process, based on a first capture
using an affinity Protein A step followed by a second step
on HEA HyperCel sorbent could successfully be used to
purify the MAb used in this study. No additional polishing
step was required as the contaminants content was
already very low after these two steps.
This approach could be used as a general tool at early
development stage to optimize the conditions to be used
on mixed-mode sorbents in any protein purification process.
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USTR 2795