Application note Reduce isoagglutinin risk while protecting yield with IsoClear® XT Robust isoagglutinin reduction with high IgG recovery across relevant process conditions.
Overview
Introduction
Isoagglutinins in IVIG are a recognized contributor to hemolysis risk and must be reduced without compromising plasma utilization, IgG recovery or process efficiency. Traditional approaches such as donor screening can restrict plasma availability while delivering only modest reductions in isoagglutinin levels. IsoClear® XT is an immunoaffinity chromatography solution designed to remove isoagglutinins while maintaining high IgG recovery.
IsoClear® A XT and IsoClear® B XT adsorbents are designed to remove isoagglutinins by immunoaffinity chromatography while maintaining high recovery of total IgG. For IVIG manufacturers, this addresses a critical challenge: reducing hemolysis risk without unnecessary compromise to plasma utilization, yield, or process efficiency 1,2.
This application note evaluates IsoClear® XT under processrelevant conditions to assess three questions that matter most to IVIG manufacturers: can isoagglutinin removal be achieved reliably, can high IgG recovery be maintained, and can the step integrate into manufacturing without creating unnecessary process burden? Across the conditions tested, IsoClear® XT delivered robust isoagglutinin reduction together with strong recovery, broad operating flexibility, and favorable processability, supporting its use as a practical polishing solution for IVIG manufacturing.
Key findings: • Achieved 2–3 titer-step isoagglutinin reduction across relevant operating conditions - Maintained high IgG recovery, averaging 96% at pH 5–5.5 and 1-minute residence time - Performed consistently across pH 4–7 and conductivity up to 30 mS/cm - Maintained effective clearance at 1-minute residence time, with additional binding margin at 5 minutes • No loss of performance after extended 0.5 M NaOH exposure • Showed robust performance across variable feedstock titers - Generated lower running pressure than a commercial comparator
Isoagglutinin reduction remains an important consideration because IVIG-associated hemolysis, although infrequent, may lead to serious clinical consequences 3-6. Regulatory expectations are also increasing, with current pharmacopeial limits criticized for not fully addressing hemolysis risk in high-dose immunomodulatory use and the EMA revising its IVIG clinical investigation guideline in 2022 to strengthen hemolysis monitoring requirements7. At the same time, plasma supply remains a major constraint for IVIG manufacturers. Donor exclusion strategies can further tighten supply while delivering only modest reductions in isoagglutinin levels, whereas immunoaffinity chromatography offers a more direct and effective route to reduction without excluding plasma donations. In practice, manufacturers need more than effective clearance alone. Any isoagglutinin reduction step must also support high IgG recovery, practical throughput, cleaning compatibility, and straightforward integration into existing downstream workflows. IsoClear® XT is designed to meet these requirements by combining robust isoagglutinin removal with strong IgG recovery and process-friendly operating characteristics. The commercial implications may also be substantial. For a large-scale fractionator processing 10 million liters of plasma per year, recovering the additional 5% of plasma typically excluded through donor screening may represent up to £120 M in additional annual product value*, before considering the added benefit of improved process recovery. In the same context, a 1% change in IVIG recovery may represent up to
£24 M* of annual product value, making yield preservation as important as isoagglutinin clearance.
Performance across relevant process conditions
IsoClear® XT is designed to help manufacturers improve product safety while protecting manufacturing value by combining robust isoagglutinin removal with high IgG recovery, supporting safer IVIG production without unnecessary compromise to throughput, yield, or plasma utilization.
1. Operating flexibility across relevant process conditions
*This application note focuses on technical performance under defined laboratory conditions. Any economic implications will depend on individual manufacturing scale, yield assumptions, and product value.
Study design To assess process fit, IsoClear® XT was evaluated across a range of operating conditions relevant to IVIG manufacturing, including pH, conductivity, residence time, feedstock titer, and NaOH exposure. This study examined: • Broad operating flexibility across pH and conductivity
IsoClear® XT achieved at least a 2-titer-step isoagglutinin reduction at pH 4–7, with the best balance of clearance and IgG recovery observed around pH 5. IsoClear® XT was evaluated across a broad pH range to determine how flexibly it could support isoagglutinin reduction while maintaining IgG recovery, a factor that can influence ease of integration, feed preparation requirements, and overall process robustness. Figure 1 shows how breakthrough timing changed across the pH screen, helping identify the operating range in which IsoClear® XT maintained stronger isoagglutinin binding. Effective clearance was observed across pH 4–7, with weaker performance at pH 9.
• Effective performance at short residence times
Flow-through fraction titer (A isoagglutinin)
• Consistent performance across variable feedstocks
256 128
• Favorable pressure-flow behavior compared with a commercial comparator
Titer (1/)
• Cleaning robustness under extended NaOH exposure
64 32 16 8 4
Across the application note, performance was assessed against practical process outcomes: isoagglutinin reduction, IgG recovery, and pressure-flow behavior. Experimental conditions are provided in the Experimental overview.
2 0
10
20
30
40
50
pH 7
pH 9
60
70
CV pH 4
pH 5
Flow-through fraction titer (B isoagglutinin) 256
Titer (1/)
128 64 32 16 8 4 2 0
10
20
30
40
50
pH 7
pH 9
60
CV pH 4
pH 5
Figure 1: Flow-through fraction agglutination titer across pH range showing A and B isoagglutinin clearance at pH 4–7. The pH 9 condition has earlier breakthrough of isoagglutinin, indicating less binding. Load titer was 1/128 A and 1/64 B isoagglutinin.
70
Non-bound isoagglutinin titer compared to load 32
32
Titer (1/)
16 8
8
8 4
4
4
2. Minimal feed adjustment and process compatibility
2
2
1
1 A isoagglutinin
1
B isoagglutinin Load
pH 4
pH 5
98
95 89
90 80
Recovery (%)
IsoClear® XT performed consistently across a conductivity range of 5–30 mS/cm.
pH 7
IgG recovery at A280 (%) 100
Overall, the data show that IsoClear® XT can maintain strong isoagglutinin reduction across a broad pH range, with the best overall balance of clearance and IgG recovery around pH 5. The weaker result at pH 9 helps define the upper boundary of the effective operating range, while the pH 7 data suggest recovery can be further tuned through wash optimization.
70
Conductivity tolerance is an important practical consideration because extensive feed adjustment adds complexity and time. IsoClear® XT was therefore assessed across a conductivity range relevant to IVIG processing to determine whether strong performance could be maintained without narrow conditioning requirements.
60 50 40 30 20 10 0 pH 4
pH 5
pH 7
Figure 2: Total non-bound agglutination titer and IgG recovery. Load titer was 1/32 for A and for 1/8 B isoagglutinin. The pH screening results are translated into a process-style pooled format in Figure 2, showing the overall titer reduction and IgG recovery in the collected non-bound fraction. Under these more process-relevant conditions, pH 5 and pH 7 delivered the strongest overall clearance, with a 3-titer-step reduction in the non-bound, while pH 4 still achieved the target reduction, with a 2-titer-step reduction in the nonbound. It is shown in Figure 3 that adding salt to the pH 7 wash improved IgG recovery while still maintaining the target 2-titer-step reduction. This highlights the potential to tune the balance between recovery and clearance where additional optimization is needed. Parameter
IsoClear® XT achieved a 2-titer-step reduction of both anti-A and anti-B isoagglutinins together with 98–99% IgG recovery across the full 5–30 mS/cm conductivity range at pH 5.5. Comparable performance was also observed at lower conductivity, suggesting that little or no conductivity adjustment may be needed before loading. Isoagglutinin reduction and IgG recovery remained essentially unchanged across the conductivity range tested, as shown in Figure 4, supporting flexible integration without narrow feed conditioning requirements.
3. Throughput and productivity IsoClear® XT achieved a 3-titer-step isoagglutinin reduction at a 1-minute residence time. Residence time directly affects throughput, making it a key parameter for process productivity. IsoClear® XT was assessed at short residence times to determine whether strong isoagglutinin clearance and high IgG recovery could be maintained under conditions compatible with efficient manufacturing.
A isoagglutinin titer
B isoagglutinin titer
IgG recovery (%)
Load
1/32
1/8
1
Non-bound 0 M NaCl in wash
1/4
1/1
89
Non-bound 0.5 M NaCl in wash
1/8
1/1
97
Figure 3: Improved IgG recovery at pH 7 with addition of 0.5 M NaCl in the wash buffer. The isoagglutinin A removal was slightly reduced from a 3-titer step to 2-titer-step removal with the higher conductivity wash buffer.
Non-bound isoagglutinin titer across conductivity range
Flow-through fractions (A isoagglutinin) at 1- or 5-minute residence time
16
16
16
4
4
4
8
Titer (1/)
8
8
Titer (1/)
(A)
4
4 2
2
2
2
1
2
0
10
20
30
40
50
60
70
80
70
80
CV 1 min
1 A isoagglutinin Load
NB 5 ms/cm
B isoagglutinin NB 15 ms/cm
(B)
NB 30 ms/cm
IgG recovery at UV A280 (%) 98
99
98
90 80
Recovery (%)
Flow-through fractions (B isoagglutinin) at 1- or 5-minute residence time
16 8
Titer (1/)
100
5 min
4 2
70
1
60
0
50
10
20
30
40
50
60
CV
40
1 min
30
(C)
20
Non-bound isoagglutinin titer at 1- or 5-minute residence time
10 0 NB 15 mS/cm
32
NB 30 mS/cm
Figure 4: Non-bound (NB) isoagglutinin titer and IgG recovery showing 2-titer-step reduction and 98–99% IgG recovery at 5–30 mS/cm. IsoClear® XT maintained meaningful clearance even at a 1-minute residence time, while the 5-minute condition provided an additional margin of binding performance, as shown in Figure 5. This supports the use of short residence times where throughput is a priority.
4. Robustness across feedstock variability IsoClear® XT achieved a 2-titer-step isoagglutinin reduction in both high- and low-titer feedstocks.
32
16
Titer (1/)
NB 5 mS/cm
5 min
8
8 4
4 2
2
1
1 A isoagglutinin Load
(D)
1
B isoagglutinin 1 min
5 min
IgG recovery by UV A280 (%)
100
94
97
90 80 70 60 50
Feedstock variability is a practical reality in plasma fractionation. To assess robustness, IsoClear® XT was evaluated using feedstocks with different starting isoagglutinin titer to determine whether target clearance could still be achieved without compromising process applicability.
40 30 20 10 0 1 minute
5 minutes
Figure 5: (A,B) Flow-through fractions show breakthrough of isoagglutinin at 1-minute residence time before 5-minute residence time. (C) Non-bound isoagglutinin clearance shows 3-titer-step reduction at 1-minute residence time and an increased removal for A isoagglutinin with 5-minute residence time. (D) Both 1-minute and 5-minute residence time show excellent IgG recovery.
Flow-through fractions low- and high-titer feedstock (A isoagglutinin)
5. Cleaning robustness As cleaning compatibility is essential for implementation, IsoClear® XT was challenged under extended NaOH exposure to assess whether performance could be maintained under caustic conditions relevant to manufacturing.
Titer (1/)
64 16 4 1
0
10
20
30
40
50
60
70
80
CV Low
High
Flow-through fractions low- and high-titer feedstock (B isoagglutinin) Titer (1/)
64 16 4 1
0
10
20
30
40
50
60
70
80
CV Low
High
NaOH robustness was first assessed in a breakthrough study after 210 hours of exposure to 0, 0.5, or 1 M NaOH. Columns were then loaded with feedstock and the flow-through collected in 1 CV fractions to compare breakthrough behavior after caustic challenge. Breakthrough behavior remained largely unchanged after extended exposure to 0.5 M NaOH, supporting compatibility with standard cleaning conditions, as covered in Figure 7. Even after the more aggressive 1 M NaOH challenge, B isoagglutinin performance was maintained and only a modest reduction in A isoagglutinin binding was observed.
Isoagglutinin removal from low- and high-titer feedstock
Flow-through fractions after NaOH exposure (A isoagglutinin)
128 128 64
32
64 32
16
16
16
16 8 8 4
4
Titer (1/)
Titer (1/)
32
8 4 2
2
2
1 0
1
Low-titer NB
20
30
B isoagglutinin High-titer load
0M
High-titer NB
Figure 6: (Top) Flow-through fractions from high- and low-titer feedstock. (Bottom) Non-bound isoagglutinin titer compared to load for low- and high-titer feedstock. The load titer was 1/128 for A and 1/64 for B isoagglutinin for the high titer and 1/16 for A and 1/8 for B isoagglutinin for the lowtiter feedstock.
40
50
60
70
80
70
80
CV 0.5 M
1M
Load
Flow-through fractions after NaOH exposure (B isoagglutinin) 32 16
Titer (1/)
A isoagglutinin Low-titer load
10
8 4 2
As shown in Figure 6, higher starting titer led to earlier breakthrough, but target clearance was still achieved at the same load volume. Where greater clearance is needed for higher-titer material, loading conditions can be further optimized.
1
0
10
20
30
40
50
60
CV 0M
0.5 M
1M
Load
Figure 7: Flow-through fractions from IsoClear® XT 50:50 blended columns after exposure to 0 M, 0.5 M, or 1 M NaOH for 210 hours.
Non-bound isoagglutinin removal after 0.5 M NaOH exposure
IgG recovery on IsoClear® XT at pH 5–5.5 from multiple batches of feedstock 100
Recovery (%)
It is confirmed in Figure 8 that extended exposure (430 hours) to 0.5 M NaOH did not affect overall isoagglutinin reduction or IgG recovery in the pooled non-bound fraction. This supports the robustness of IsoClear® XT under prolonged caustic exposure relevant to cleaning-in-place strategies.
98
95
96
95
94
94
94
5
6
92
32
32
99 98
98
90 0
Titer (1/)
16 8 4
4
4
1
1 A isoagglutinin Load
Control (0 M)
1
B isoagglutinin
-95% CI
105 95
90 80 70 60
40
20 10 0 0.5 M NaOH exposure
Figure 8: Isoagglutinin removal and IgG recovery of 50:50 blended IsoClear® XT after 430 hours exposure to 0.5 M NaOH.
6. Recovery consistency IsoClear® XT achieved consistently high IgG recovery at approximately pH 5 with a 1-minute residence time. Recovery consistency was reviewed from two perspectives: average recovery across multiple feedstocks and repeatability across multiple cycles using the same feedstock. Together, these data show consistently strong recovery under processrelevant conditions, with an average of 96% ± 1.8% (t95%, n = 7, Figure 9). As illustrated in Figure 10, IgG recovery remained highly consistent across seven repeated cycles using the same feedstock. This repeatability supports reliable operation under standard process and cleaning conditions.
8
103 101
101
100 99
99
98
99 97
0
1
2 IgG recovery
30
7 +95% CI
103
95
50
Control (0 M)
4 Average
IgG recovery on IsoClear® XT with the same feedstock at pH 5.5 and 1-minute residence time
0.5 M NaOH exposure
Recovery (%)
94
3
Figure 9: IgG recovery from experiments outlined above which were run at pH 5 to 5.5 with 1-minute residence time, average recovery was 96% ± 1.8% (t95%, n = 7 confidence interval).
IgG recovery by UV A280 (%) 100
2 IgG recovery
2
Recovery (%)
1
8
3
4 Average
5 -95% CI
6
7
8
+95% CI
Figure 10: IgG recovery from running the same batch of feedstock at pH 5.5 with 1-minute residence time, average recovery was 101% ± 2.3% (t95%, n = 7 confidence interval).
7. Pressure-flow performance IsoClear® XT generated lower running pressure than a commercial comparator, supporting easier process handling and scale-up. Pressure-flow behavior matters because it affects processability, operating flexibility, and scale-up. Testing was carried out using blended A and B resins under the same conditions. In this study, IsoClear® XT generated lower running pressure than a commercial comparator. The pressure advantage of IsoClear® XT relative to a commercial comparator is shown in Figure 11, illustrating lower running pressure across the tested flow range. In practice, this lower back pressure can simplify operation and provide greater headroom when increasing flow rate or moving to larger formats.
DeltaC pressure between IsoClear® XT and comparator
Experimental overview
DeltaC pressure (MPa)
0.12
Feedstock
0.1 0.08
IsoClear® XT was evaluated under process-relevant conditions designed to reflect IVIG manufacturing workflows.
0.06 0.04 0.02 0 0
200
400
600
800
1000
1200
cm/hr Comparator
IsoClear® XT
Figure 11: Delta column pressures of IsoClear® XT compared to a 50 µm commercially available adsorbent.
Conclusions IsoClear® XT gives IVIG manufacturers a practical way to reduce isoagglutinin risk without giving up the yield and process performance that drive manufacturing value. Across the conditions evaluated, it consistently combined effective isoagglutinin reduction with high IgG recovery, broad operating flexibility, and favorable processability. The study showed that IsoClear® XT delivered 2–3-titer-step isoagglutinin reduction while maintaining strong IgG recovery across relevant operating conditions, including short residence time, variable feedstock titer, extended 0.5 M NaOH exposure, and a broad pH and conductivity range. Together, these data support IsoClear® XT as a robust polishing step that can be integrated into IVIG workflows without unnecessary process burden. For manufacturers, this means product safety can be improved while protecting plasma utilization, throughput and yield. Combined with lower running pressure than a commercial comparator, IsoClear® XT offers a flexible, process-friendly solution for safer and more efficient IVIG manufacturing.
Condition
An IVIG intermediate feedstock was prepared to represent a relevant process stream for evaluation of IsoClear® XT. The material was derived from human plasma using MiMode PuraBead® HL3 adsorbent, followed by a precipitation step and buffer exchange into 10 mM sodium acetate, pH 5.2. This preparation removed the majority of albumin and IgM impurities while retaining 94% of the IgG. For pH-screening studies, the feedstock was adjusted to the target pH and matched to the corresponding equilibration buffer before loading (Table 1). For conductivity studies, equilibration buffers at 5, 15, and 30 mS/cm were prepared using 10 mM sodium acetate, pH 5.5, supplemented with NaCl. The feedstock was adjusted to match the conductivity of the corresponding equilibration buffer before loading.
Column operation IsoClear® A XT and IsoClear® B XT were blended 50:50 and packed into 10 × 125 mm SNAP® columns, giving a 1.26 mL bed volume. Columns were operated in flow-through mode, with isoagglutinins binding to the adsorbent while IgG was recovered in the non-bound fraction. Unless otherwise stated, columns were equilibrated with 10 column volumes (CV) of 20 mM sodium acetate, pH 5.5, adjusted to 1.3 mS/cm to match the feedstock conductivity. Columns were then loaded with IVIG intermediate, typically 63–115 CV depending on the experiment and washed with equilibration buffer until the UV signal returned to baseline. For breakthrough studies, 1 CV flow-through fractions were collected across the load. For process-representative studies, the full flow-through and post-load wash were pooled to determine overall isoagglutinin reduction and IgG recovery.
Feedstock final pH and conductivity
Equilibration buffer
pH 4
pH 4.03, 1.16 mS/cm
20 mM sodium acetate, pH 4 1
pH 5
pH 5.07, 2.02 mS/cm
20 mM sodium acetate, pH 5
pH 7
pH 7.04, 3.37 mS/cm
20 mM sodium phosphate, pH 7
pH 9
pH 9.00, 1.57 mS/cm
20 mM Tris, pH 9
Table 1: pH adjustment of feedstock.
Where increased salt was evaluated to improve recovery, the post-load wash buffer was changed to 20 mM sodium phosphate, 0.5 M NaCl, pH 7. For the residence-time study, two experimental formats were used. In the first, flow-through was collected in 1 CV fractions to assess breakthrough, using a load titer of 1/16 for anti-A and 1/8 for anti-B isoagglutinin. In the second, the flow-through and post-load wash were pooled to assess overall process performance, using a load titer of 1/32 for anti-A and 1/8 for anti-B isoagglutinin. Between runs, columns were cleaned with 5 CV of 0.5 M NaOH with a 10-minute hold, followed by re-equilibration until the column pH matched the equilibration buffer.
Analytics and performance targets IgG recovery was estimated from UV absorbance at 280 nm (A280). Isoagglutinin levels (anti-A and anti-B) were determined by direct agglutination assay. Unless otherwise stated, successful performance was defined as at least a 2-titer-step reduction in isoagglutinins together with >90% IgG recovery.
(4) Dantal J. (2013). Adverse effects of immunoglobulin G therapy: thromboembolism and haemolysis. Clinical and Experimental Immunology, 178(Suppl 1): 72–74. PMC4285498. (5) Siani B et al. (2014). Isoagglutinin reduction in human immunoglobulin products by donor screening. Biol Ther 4:15–26. PMC4254866 (6) Tong TN et al. (2020). Treatment-associated haemolysis in Kawasaki disease: association with blood-group antibody titres in IVIG products. Blood Adv 4:3416–26 (7) EMA/CHMP/BPWP/94033/2007 rev. 4. Guideline on the clinical investigation of human normal immunoglobulin for intravenous administration. Effective 01/01/2022 For more information about MiMode® PuraBead® HL3 adsorbent, IsoClear® XT adsorbents, SNAP® columns, and PuraBead® base matrix, please visit the Astrea Bioseparations website.
References (1) Shebl, A., et al. (2020), Isoagglutinin reduction in intravenous immunoglobulin (IgPro10, Privigen) by specific immunoaffinity chromatography reduces its reporting rates of hemolytic reactions: an analysis of spontaneous adverse event reports. Transfusion, 60: 1278-1286. (2) Gerber, S. et al (2016) Reduction of Isoagglutinin in Intravenous Immunoglobulin (IVIG) Using Blood Group Aand B-Specific Immunoaffinity Chromatography: IndustryScale Assessment. BioDrugs 30, 441–451 (2016). https:// doi.org/10.1007/s40259-016-0192-3 (3) Gaines AR. Disseminated intravascular coagulation associated with acute hemoglobinemia or hemoglobinuria following Rh(0)(D) immune globulin intravenous administration for immune thrombocytopenic purpura. Blood. 2005 — but more directly the language about 1 in 10,000 and deaths comes from:
Astrea Bioseparations is a world class provider of chromatography adsorbent and resin services. With over 30 years of chromatography manufacturing expertise, we deliver a unique and trusted service in close partnership with our clients. For more information, please don’t hesitate to reach out at sales@astrea-bio.com or visit astreabioseparations.com. Any data or results provided are only examples and do not provide any guarantee of similar results in future. The products of Astrea Bioseparations may be covered by or for use under one or more patents: astreabioseparations.com/patents All trademarks, trade names, trade dress, product names and logos are the property of Astrea UK Services Ltd or their respective owners. @2026 Astrea Bioseparations Ltd. All rights reserved.