Removal of Isoagglutinins from IVIG and Plasma Using Affinity Chromatography Bastiaan Lobbezoo, Lucy Pepperell, Patrick V. Gurgel, Steve Burton, Astrea Bioseparations Ltd, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
80
• Passive transfer of IgG and anti-A and Anti-B isoagglutinins play a role in those events
Astrea Bioseparations developed affinity chromatography resins for the removal of isoagglutinins from plasma and plasma-derived products, such as IVIG. The resins, IsoClear A and IsoClear B, can clear isoagglutinins from a titre of 1/32 down to neat (no observable agglutination) using a load of up to 50 CV of plasma. A column of IsoClear A loaded with 200 CV of O plasma can reduce the titre of isoagglutinin A from 1/32 to neat using a 3-minute residence time. When used in the production of IVIG, the resins can achieve a reduction of A-isoagglutinins from 1/14 to 1/4 and B-isoagglutinins from 1/6 to neat when loading 70 CV of an IVIG process intermediate. The resins have also been evaluated as a blend, allowing the removal of both A- and B-isoagglutinins in a single step. Typical values for IgG recovery are > 90%.
68.4
50.3
50
1:8
50.3
50
47.5
40 30
26.5
20 10
5.2 1:8
1:4
B
61.1
60
% of lots at titer level by plasma type
% of lots at titer level by plasma type
68.4
70 60
2.3
54.8
50 38.1
40
30.7
30 20 10
7.5
70
Reactions with donor’s red blood cells
ABO Antibodies
ABO blood type
None
Anti-A Anti-B
O
A
Anti-B
A
B
Anti-A
B
• Cause agglutination of red blood cells • Growing concern for transfusion and plasmaderived products
Donor type O cells
Donor type A cells
Donor type B cells
% of lots at titer level by plasma type
Recipient’s blood
B
1:1
A&B
61.1
60
Solution: Removal of isoagglutinins using affinity resins IsoClear A and IsoClear B 38.1
40
30.7
30 20 10
7.5 0.2
0
None
1:1
5.4
1.7 1:2
Resin Re-use
AB
0.6 1:4
1:8
1:16
Sample
Compatible
Non-compatible
Recovery of IgG
IsoClear B Trisaccharide
For the capture of anti-A isoagglutinins
Ligand structure GalNAc
For the capture of anti-B isoagglutinins
A
A
White microspheres
MEAN PARTICLE SIZE:
65 µm
MEAN PORE DIAMETER:
1000 Ångström
MATRIX:
HO
-
1/32
Run 1
78.0%
1 (Neat)
Run 4
99.8%
1 (Neat)
Run 41
97.4%
1 (Neat)
Run 51
102.9%
1 (Neat)
Activate
Resins are fully validated, produced at up to 50-L batch sizes in a GMP facility
O
OH OH
O O
OH
HO
2-mL column of IsoClear B using A plasma. 50 CV of plasma loaded per run (3-min RT), followed by a post-load wash, elution, and CIP with 20% ethanol, 1 M Acetic acid.
At least 51 cycles when using plasma
Sample
A Isoagglutinin
B Isoagglutinin
Load
1/14
1/6
Non-bound
1/4
Neat
Elution
1/64
1/32
CIP
1/128
1/32
• Protein recoveries of up to 99.7% • Full recovery of coagulation factors
1/16
1/32
X
-NH-Antigen
Antigen-NH2
1
2
3
97
Clearance of Anti-A Isoagglutinins from O-plasma using IsoClear A
Non-bound
4
5
• Guanidine salts show good resin cleaning • Inclusion of a reducing agent in the CIP aids in the removal of tightly bound proteins. • TCEP is an alternative to DTT, at lower concentrations
Efficient cleaning regimes
64
51
39
28 19 14 2-mL IsoClear A column loaded with 200 CV of plasma (3 min retention time)
1- MW marker 2- Control (20% Ethanol/1 M acetic acid) adsorbent 3- Guanidine hydrochloride adsorbent 4- Guanidine thiocyanate adsorbent 5- Guanidine carbonate adsorbent
Clearance of Anti-B Isoagglutinins from A-plasma using IsoClear B
• Full recovery of antibodies • Full recovery of TTP factors
Conclusions
• Efficient recovery of isoagglutinins from plasma and IVIG process intermediates • Can be used as a blend IsoClear A + IsoClear B 2-mL IsoClear B column loaded with 300 CV of plasma (3 min retention time)
Plasma Product Biotechnology 2017 – Malta | Trademark Information. © Astrea Bioseparations Ltd 2017. All rights reserved
1/128
Demonstration of Isoagglutinin clearance from plasma using IsoClear B. Removal of 1/64 down to 1/2 (dilution at where there is no observable agglutination)
Resin CIP Study
Removal from plasma
Search: Astrea Bioseparations
1/64
-NH-Antigen -NH-Antigen
191
Removal from IVIG process intermediate
1/8
Antigen-NH2Couple
kDa
Resin Performance
1/4
Antigen-NH2
OH (Porous resin beads)
X
OH
OH
NH O OH Ac OH O Ac HO HO O HO OH O HO O HO O
HO
X
O
O O
1/2
Load
OH
OH
NH
α 1-2
(Porous resin beads)
O NH
Couple Couple
OH Ligand attachment (Porous resin beads)
2 – 30 °C, 20% ethanol
HO HO O
O
Ac
α 1-3 Gal β1 Activate Activate
FucOH
Up to 1 bar
STORAGE CONDITIONS:
1-2 αα 1-2
Fuc Fuc
B
HO
HO
α 1-3
Gal β1 α 1-3 Gal β1 α Gal 1-2 β1 -
Gal
Fuc
Gal
Up to 500 cm/h
RECOMMENDED OPERATING PRESSURE:
A B B
HO
α 1-3
Gal
OH
HO
β1 αGal1-2
α 1-2
OH
HO
α 1-3Gal β1 -
Fuc GalNAc
Pack at flow rates up to 850 cm/h (pressure up to 2 bar) using 0.1 M NaCl solution
RECOMMENDED OPERATIONAL FLOW RATE:
GalNAc
Fuc
Beaded polymethacrylate resin
RECOMMENDED PACKING CONDITIONS:
α 1-3
Agglutination
Load
Neat
IsoClear A
LIGAND:
ADSORBENT APPEARANCE:
1:16
54.8 (A) Anti-A and (B) anti-B titer levels in finished IVIG products. Purple bars refer to recovered plasma, and yellow50bars are source plasma. From McVey et al., Transfusion, March 2015.
Donor type AB cells
Resin Properties FUNCTION:
0.6 1:8
1:4
1:2
Anti-B Titer Dilution Levels
Anti-B Titer Dilution Levels
• AB plasma is devoid of isoagglutinins, and can be used by all blood type recipients. A, B, and O-type bloods may contain isoagglutinins
RESIN
5.4
1.7
0.2
0
1:16
Anti-A Titer Dilution Levels
ABO antigens
1:16
Anti-A Titer Dilution Levels 70
A
0
• Isoagglutinins are isoantibodies normally present in serum
26.5
20
• P10roducts with a median anti-A equal or lower than 1:16 5.2 2.3 are associated with lower haemolytic risk (EUDRA) 0 1:4
80
The resins use a polymethacrylate base matrix, and can be reused for at least 50 cycles with no loss of functionality, using a CIP process not involving NaOH, due to the sensitivity of the affinity ligands to those conditions. The resins offer excellent physical stability, with good flow properties.
Isoagglutinins
47.5
40
• M30aximum allowable limit is 1:64 (Ph. Eur. and WHO)
• High-dose IVIG treatment implicated in haemolytic events
Antibody-mediated Haemolysis is a hard to predict phenomenon with potentially severe consequences. It is mediated by naturally-occurring anti-A and anti-B immunoglobulin isoagglutinins, which are present in plasma, blood, and several derived products, including IVIG produced by plasma fractionation.
% of lots at titer level by plasma type
Potential Problems
Abstract
A
70 60
• Efficient removal of isoagglutinins from complex mixtures • High degree of protein recovery • More than 50 re-use cycles • Efficient CIP using guanidine and DTT or TCEP