Application Note
New Streamlined IgG Purification Process
From Human Plasma Using S HyperCel™
and HyperCel STAR AX Ion Exchange
Chromatography Sorbents
USD 2903(1)
1.
Introduction
Current IVIG purification processes often combine precipitation techniques and chromatographic
separation. Even when the final IVIG purity is high, however, traces of contaminants such as IgA, or
IgM can cause severe adverse events, including anaphylactic shock [1,2].
A new IVIG process was developed combining precipitation of cryo-poor plasma using caprylic acid,
followed by ion exchange sorbent chromatography. Precipitation with caprylic acid is easily achieved at
laboratory scale and the resulting fraction contains close to physiological proportions of IgG, IgA, and IgM
in plasma. This, however, represents a worst-case scenario in terms of contamination levels of IgA and
IgM, compared to intermediate plasma fractions. Additionally, caprylic acid is increasingly used as an
alternative to standard ethanol precipitation, to produce human IgG products [3]. The ability of S HyperCel
sorbent (cation exchange) and HyperCel STAR AX sorbent (salt tolerant anion exchange) to remove IgA
and IgM was tested under a variety of process-relevant conditions of pH and conductivity.
A structured experimental design (DoE – Design of Experiments) was applied, combined with fast
screening in AcroPrep® Advance 96-well filter plates and PRC prepacked columns. This design approach
gave a broader understanding of operational limits and reduced development time. Extensive removal of
IgA (to <0.5%) and IgM (to undetectable levels) was achieved with high yield (>85%) of recovery with
minimal impact on IgG sub-class distribution (less than 20% loss of IgG4) after a two-step chromatography
process.
This work was performed in collaboration with Dr. Thierry Burnouf from Human Protein Process Sciences
(HPPS) and gave rise to a publication in the journal Transfusion [4].
2.
Material and Methods
The strategy for the development of a two-step purification for human plasma IgG used a combination
of (i) DoEs and high throughput 96-well chromatography filter plates, and (ii) on-column chromatography
experiments (Figure 1).
Starting Plasma Feedstock
Frozen crude plasma was thawed at 4 °C to precipitate most clotting factors (cryo-precipitation). The
thawed sample was then centrifuged and the supernatant filtered through a 0.2 μm filter. The cryo-poor
plasma, was then further processed to remove non-antibody proteins using precipitation by caprylic acid
(5% v/v, pH 5.5).
Optimization of Operating Conditions
Condition ranges applied for optimization of conditions were as described in Figure 1. A first set of
experiments (DoE 1) was conducted using AcroPrep Advance 96-well filter plates filled with S HyperCel
sorbent to define pH and conductivity loading conditions for the first chromatography step in order to
remove IgA and IgM contaminants while capturing IgG efficiently. The total immunoglobulin load was set
to 5 mg/mL of sorbent. As the loading conditions were defined, the best elution conditions were identified
using data from a second set of experiments (DoE 2) using 96-well filter plates handled in the same
conditions.
2
Transfer to Column
The selected pH and conductivity conditions for binding and elution on S HyperCel sorbent were then
transferred on a 5 mL Pall PRC prepacked column.
The elution pool from the S HyperCel PRC prepacked column was finally directly loaded on a 1 mL
HyperCel STAR AX PRC prepacked column for the second orthogonal purification step. The amount of
immunoglobulin loaded on the HyperCel STAR AX PRC column was defined to maximize IgG yield of
recovery in the flow through (FT) while keeping IgA and IgM captured.
Analytical Methods
ELISA assays were used to specifically quantify IgG, IgA, IgM in the initial sample (supernatant from
caprylic acid precipitation), FT and elution fractions from the S HyperCel PRC column, and FT fractions from
the HyperCel STAR AX PRC column. IgG sub-classes 1 to 4 were also analyzed using specific ELISA assay.
Figure 1
Strategy for the Development of a Two-Step IgG Purification Process from Crude Plasma
Cryo-precipitate
Cryo-precipitation
High Throughput 96-Well
Plate Chromatography
Cryo-poor plasma
Caprylic acid
precipitate
Caprylic acid
precipitation
Caprylic acid
supernatant
pH 5.5, 13 mS/cm
DoE 1:
Load pH/conductivity on CEX
(S HyperCel sorbent)
pH 4.0 – 5.5, 2 – 13 mS/cm
DoE 2:
Elution pH/conductivity on CEX
(S HyperCel sorbent)
pH 6.0 – 8.0, 2 – 20 mS/cm
........................................................................
Plasma
........................................................................
Pre-Chromatography
Processing
Chromatography
on Column
CEX bind/ elute mode
(S HyperCel sorbent)
Load pH 4.5, 8 mS/cm
Elution pH 8.0, 12 mS/cm
AEX FT mode
(HyperCel STAR AX sorbent)
Load pH 8.0, 12 mS/cm
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3.
Results
3.1.
Selection of Loading Conditions on S HyperCel Sorbent Using DoE and 96-Well Filter
Plates
Figure 2
Selection of Loading Conditions on S HyperCel Sorbent
IgG
10
8
6
4.75
5.00
pH
5.25
12
10
8
6
4
4.50
5.50
4.75
13
5.00
pH
5.25
5.50
IgG, IgA, IgM
in FT (% of load)
12
10
8
6
4
4.50
4.75
5.00
pH
5.25
5.50
< 10
10 – 20
20 – 30
30 – 40
40 – 50
50 – 60
60 – 70
70 – 80
80 – 90
> 90
IgA in FT (%)
20
100
12
11
Conductivity (mS/cm)
IgM
Conductivity (mS/cm)
12
4
4.50
B
IgA
Conductivity (mS/cm)
Conductivity (mS/cm)
A
IgM in FT (%)
20
100
10
9
IgG in FT (%)
0
15
8
7
6
5
4
4.6
4.7
4.8
4.9
5.0
5.1
5.2
5.3
5.4
5.5
pH
A: Contour plots from response surface modeling of DoE 1: % of loaded IgG, IgA, IgM recovered in flow
through (FT) vs. loading pH and conductivity. B: Sweet spot loading conditions for IgG in FT <15%, IgA and
IgM in FT >20%
Best loading conditions on S HyperCel sorbent to maximize IgG binding while removing IgA and
IgM in the FT were determined from the screening of loading conditions (DoE 1) (Figure 2). Loading
at low pH and low conductivity appeared as essential for good IgG binding (very low % in the FT
fraction). In contrary, high loading pH and conductivity resulted in high IgA and IgM removal (high %
in the FT fraction) (Figure 2A). In order to accommodate high IgG binding and satisfying removal of
IgA and IgM, a fairly large operational design space was determined (Figure 2B) where limiting loss
of IgG and sufficient removal of IgA and IgM in the FT fraction were obtained. Therefore, optimum
loading conditions on S HyperCel sorbent were selected accordingly at pH 4.5 and conductivity
8 mS/cm.
4
Selection of Elution Conditions on S HyperCel Sorbent Using DoE and 96-Well Filter
Plates
Figure 3
Selection of Elution Conditions on S HyperCel Sorbent
IgG
IgA
15
10
5
5.0
5.5
6.0
6.5
pH
7.0
7.5
20
Conductivity (mS/cm)
Conductivity (mS/cm)
IgM
20
IgG in elution
(% of load)
< 10
10 – 20
20 – 30
30 – 40
40 – 50
50 – 60
60 – 70
> 70
Conductivity (mS/cm)
20
A
15
10
5
8.0
5.0
IgA, IgM in
elution (% of load)
15
5
10
15
20
25
10
5
5.5
6.0
6.5
pH
7.0
7.5
8.0
5.0
5.5
6.0
6.5
pH
7.0
7.5
<5
– 10
– 15
– 20
– 25
– 30
> 30
8.0
20
B
IgA in elution (%)
0
10
15
Conductivity (mS/cm)
3.2.
IgM in elution (%)
0
50
IgG in elution (%)
70
100
10
5
5.0
5.5
6.0
6.5
pH
7.0
7.5
8.0
A: Contour plots from response surface modeling of DoE 2: % of bound IgG, IgA, IgM recovered in elution vs.
elution pH and conductivity. B: Sweet spot elution conditions for >70% IgG recovery, <10% IgA in elution and
<50% IgM in elution.
Best elution conditions on S HyperCel sorbent to obtain high IgG yield of recovery and good
contaminant (IgA and IgM) removal were determined after loading the sample at pH 4.5 and
8 mS/cm as defined from the first set of high throughput experiments (Figure 3). The highest yield
of recovery for IgG was obtained at pH and conductivity above 7.0 and 10 mS/cm respectively.
A good IgA elimination (<10% in elution fraction) was achieved independently of the conditions
used for elution, but IgM removal was more challenging since conditions to obtain less than 10%
IgM in elution would not allow good IgG recovery (<40%) (Figure 3A). Conditions to elute from S
HyperCel sorbent (pH 8.0, conductivity 12 mS/cm) were therefore selected based on IgG recovery
(Figure 3B).
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3.3.
On-Column Two-Step IgG Purification on S HyperCel and HyperCel STAR AX Sorbents
Operating conditions to be used on S HyperCel sorbent determined from 96-well filter plate
experiments were successfully transferred on a 5 mL S HyperCel PRC prepacked column. The
dynamic binding capacity (DBC) for IgG at 0% breakthrough using the defined loading conditions
(pH 4.5, 8 mS/cm) was first measured (37.5 mg/mL of sorbent) and the operational binding capacity,
corresponding to 80% of this DBC, was set (30 mg/mL of sorbent). The complete run on S HyperCel
sorbent was then performed using defined capacity, loading and elution pH and conductivity
(Figure 4).
Figure 4
Chromatogram of IgG Capture Run on 5 mL S HyperCel PRC Prepacked Column in Bind/Elute Mode
mAU
pH
UV 280 nm
pH
Cond (mS/cm)
>95% pure IgG
2000
10.0
1500
8.0
1000
6.0
Injection
500
.
0
0
100
200
300
mL
Equilibration and wash: 25 mM Na acetate, pH 4.5, 8 mS/cm. Load: caprylic acid supernatant adjusted to
equilibration conditions (150 mg IgG). Elution: 25 mM Tris-HCl, pH 8.0, 12 mS/cm. Strip: 25 mM Tris-HCl,
pH 8.0, 1 M NaCl. Residence time: 2 minutes
Elution fraction from S HyperCel sorbent (pH 8.0, 12 mS/cm) was directly loaded on 1 mL HyperCel
STAR AX PRC prepacked column. The optimum load of immunoglobulin to keep IgG purity above
99.5% was measured at 125 mg/mL of sorbent (Figure 5).
Figure 5
Chromatogram of IgG Purification on 1 mL HyperCel STAR AX PRC Prepacked Column in Flow
Through Mode
pH
UV 280 nm
pH
Cond (mS/cm)
mAU
10.0
800
>99.5% pure IgG
600
8.0
400
6.0
200
4.0
0
0
20
40
60
80
100 mL
Equilibration and wash: pH 8.0, 12 mS/cm. Load: post S HyperCel sorbent elution at pH 8.0, 12 mS/cm
(125 mg IgG). Strip: pH 4.0, 1 M NaCl. Residence time: 2 minutes
Supernatant from caprylic acid precipitation (initial load sample), elution fraction from S HyperCel
sorbent and FT fraction from HyperCel STAR AX sorbent were analyzed for content of IgA, IgM,
total IgG and IgG sub-classes 1 to 4 to assess the final performance of the process (Table 1).
The IgG yield of recovery is over 90% for each step of the purification process. The overall IgG yield
6
of recovery for the two-step process is 85%. This level of IgG is significantly higher than what is
typically achieved in industry as current plasma fractionation processes only provide 45 to 55% IgG
yield [4]. The final IgG purity is over 99.5 %. The final IgA content is below 0.5% and final IgM content is below detection limit. The IgG sub-classes heterogeneity is maintained with a very
limited reduction for IgG4.
Table 1
Performance of IgG Two-Step Purification Process Regarding IgG Recovery, IgA and IgM Clearance,
and IgG Sub-Classes Distribution
IgG Load
(mg/mL
Sorbent)
IgG
Recovery
(% Load)
IgG
IgA
IgM
IgG1
IgG2
IgG3
IgG4
Caprylic acid supernatant
—
—
79.7
16.6
3.7
69
19
2
9
S HyperCel sorbent
30
92
96.8
2.4
0.8
70
19
2
8
HyperCel STAR AX sorbent
125
92
99.8
0.2
0
70
19
2
8
% Total Ig
% Total IgG Sub-Classes
The next generation two-step purification process proposed here allows an efficient and streamlined
purification of IVIG (Figure 6A). High capacity obtained on both S HyperCel and HyperCel STAR AX
sorbents at high flow rate (2 minutes residence time on both steps), and limited sample adjustment
between those two chromatography steps, due to the salt tolerance of HyperCel STAR AX sorbent,
would lead to reduced column sizes and time to process at manufacturing scale (Figure 6B).
Figure 6
Next Generation IVIG Purification Process Proposed (A) and Anticipated Columns Set-Up at
Manufacturing Scale (B)
A
B
Plasma
Cryo-precipitation
Cryo-poor plasma
Caprylic acid precipitate
Cryo-precipitation
Caprylic acid precipitation
Caprylic acid precipitation
Caprylic acid supernatant
CEX bind /elute mode
S HyperCel sorbent
Processing Time: 2 days
Cryo-precipitate
Plasma
5,000 L
AEX FT mode
HyperCel STAR AX sorbent
Pure IVIG
60 cm I.D., 20 cm bed height
Residence time: 2.5 min
Linear flow rate: 480 cm/hr
4 cycles
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7
4.
Conclusion
u
Efficient and streamlined process developed for IgG purification from crude plasma (Figure 6)
u
Combination of caprylic acid precipitation and two chromatography steps on S HyperCel sorbent
(bind and elute mode) and HyperCel STAR AX sorbent (flow through mode) allows:
l
High IgG yield of recovery (>85% total) for the two chromatography step process. The IgG yield of
recovery is above 90% for each step
l
Very high IgG purity (>99.5%). The IgA content is below 0.5% and IgM is undetectable by ELISA
l
IgG sub-class distribution is maintained
l
Minimal sample adjustment between the two chromatography steps due to salt tolerance of
HyperCel STAR AX sorbent
l
High capacity at high flow rate on both sorbents which reduces column size and time to process at
manufacturing scale
References
[1] Carbone, J., Adverse reactions and pathogen safety of intravenous immunoglobulin. Curr. Drug Saf. 2 (2007) 9-18
[2] Katz, U., Achiron, A., Sherer, Y., and Shoenfeld, Y., Safety of intravenous immunoglobulin (IVIG) therapy. Autoimmun.
Rev. 6 (2007) 257-9
[3] Radosevich, M., Burnouf, T., Intravenous immunoglobulin G: Trends in Production Methods, Quality Control and Quality
Assurance. Vox Sang 98 (2010) 12-28
[4] Wu, Y.-W., Champagne, J., Toueille, M., Gantier, R. and Burnouf, T., Dedicated Removal of Immunoglobulin (Ig)A, IgM,
and Factor (F)XI/Activated FXI from Human Plasma IgG. Transfusion (2013), doi: 10.1111/trf.12243
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