Optimising IVIG Manufacturing Around Established
Precipitation Techniques
Yu-Wen Wu1, Jérôme Champagne2, Magali Toueille2, Rob Noel3, René Gantier4, Thierry Burnouf1,5
1Institute of Medical Biomaterials and Tissue Engineering, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 2Pall Life Sciences, Cergy, France
3Pall Life Sciences, Portsmouth, UK 4Pall Life Sciences, Westborough, MA, USA 5Human Protein Process Sciences (HPPS), Lille, France
INTRODUCTION
KEY AIMS
Plasma-derived IVIG purification processes combine precipitation
techniques and chromatographic separation, providing:
KEY RESULTS
► Development of a very efficient IVIG process
► Efficient and streamlined process developed for IVIG purification:
► Higher IVIG yield and purity than ethanol fractionation only
► Keep combination of precipitation and ion exchange
chromatography techniques
► Cost effective contaminant removal through precipitation
► Improved IVIG yield of recovery and purity
– Combined caprylic acid precipitation and two CEX/AEX
chromatography steps
– High IgG yield of recovery (> 85% total)
– High IgG final purity (> 99.5%; IgA < 0.5%; IgM and Factor
XI undetectable)
– No significant loss of any IgG sub-class
– No UF/DF nor hold step between CEX and AEX steps
► High reduction level of IgA, IgM and Factor XI
However:
► Trace of contaminants (IgA, IgM or Factor XI) may remain with
potential for adverse events (e.g., anaphylactic shock1,2 or
thrombogenicity3)
► Maintain IgG sub-class distribution
► Minimal number of hold steps/buffer exchanges
► Additional step on Mustang® S membrane adsorber can
provide robust Factor XI removal to processes starting with
Fraction II + III
STRATEGY FOR PROCESS DEVELOPMENT
Cryo-precipitation
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
Spiking Factor XI, 1 IU/mL
........................................................ ........................................
High Throughput 96-Well
Plate Chromatography
Plasma
Cryo-precipitate
........................................................ ........................................
Pre-Chromatography
Processing
Chromatography
on Column / Capsule
Yield and Contaminant Removal
Post-caprylic acid
Post-S HyperCel sorbent
Post-HyperCel STAR AX
sorbent
IgG
Recovery
(% load)
–
92
92
% Total Ig
IgG
79.7
96.8
99.8
IgA
16.6
2.4
0.2
IgG3
2
2
2
IgG4
9
8
8
IgM
3.7
0.8
BDL
BDL: Below Detection Limit
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
Post-caprylic acid
Post-S HyperCel sorbent
Post-HyperCel STAR AX
sorbent
% Total IgG Sub-Classes
IgG1
IgG2
69
19
70
19
70
19
IgG sub-class distribution more closely resembles plasma than other published IVIG distributions. A possible explanation is the different binding
mechanism associated with the HyperCel STAR AX adsorbent compared to DEAE- or Q- adsorbents.
CEX bind / elute mode
(S HyperCel sorbent)
Load pH 4.5, 8 mS/cm
Elution pH 8.0, 12 mS/cm
DoE 3:
Load pH and conductivity
(Mustang® S capsule)
Load pH 6.0 – 8.5
Load cond. 3 – 30 mS/cm
IgG Load
(mg/mL
sorbent)
–
30
125
AEX FT mode
(HyperCel STAR AX sorbent)
Load pH 8.0, 12 mS/cm
Post-Mustang S Capsule
IgG Load
(mg/mL
sorbent)
55
IgG
Recovery
(% load)
95
Factor XI
(ng/mL)
4.7
Factor XI
Reduction
(fold)
26
Adjustment pH 6.0
12.7 mS/cm
CEX FT mode
(Mustang S capsule)
Load pH 6.0, 12.7 mS/cm
PROPOSED IVIG PURIFICATION PROCESS STREAMS
A: Combined caprylic acid precipitation and streamlined CEX/AEX chromatography steps
B: Mustang S capsule option to remove FXI from II + III fraction
Implication for Process Scale-up (5000 L batch,
starting with Fraction II+III paste)*
A
B
Fraction II + III paste containing 75 kg IgG
Plasma
Cohn fractionation
Extraction tank
1. Carbone, J., Adverse reactions and
pathogen safety of intravenous Immunoglobulin. Curr. Drug Saf. 2 (2007) 9-18.
7000–8000L
pH 4.0–6.0 + Filter aid
DAY 1
Seitz® Supra 80P Depth Filtration
Cryoprecipitate
Cryoprecipitation
Fraction II + III
Caprylic acid
supernatant
CEX bind /elute mode
S HyperCel sorbent
AEX FT mode
HyperCel STAR AX
sorbent
DAY 2
Caprylic acid
precipitation
Pure IVIG
3. Wolberg, A.S., et al., Am. J. Hematol.
65 (2000) 30-34.
S HyperCel™ Sorbent
140 cm diameter column, volume 270 L
pH 4.0–6.0 load
450 cm/hr, 9 cycles
elution pH 8.0, 12 mS/cm
HyperCel STAR AX Sorbent
100 cm diameter column, volume 110 L
Load = S HyperCel
elution (flow through)
400 cm/hr, 5 cycles
In-process pH adjust from 8.0 to 6.0
Bioburden Reduction on Supor EAV or ECV Membrane
Mustang® S Capsules
Parallel /series configuration
CEX bind /elute mode
S HyperCel sorbent
Flow through
10 MV/min
Ultipor® DVD Virus Prefilter
CEX FT mode
Mustang S capsule
Pure IVIG
DAY 4
AEX FT mode
HyperCel STAR AX
sorbent
2. Katz, U., Achiron, A., Sherer, Y., and
Shoenfeld, Y., Safety of intravenous
immunoglobulin (IVIG) therapy.
Autoimmun. Rev. 6 (2007) 257-9.
Bioburden Reduction on Supor EAV or ECV Membrane
DAY 3
Caprylic acid
precipitate
Bioburden Reduction on Supor EAV or ECV Membrane
S/D or low pH incubation tank
Cryo-poor plasma
References
Pegasus™ SV4 Virus Filter Cartridge
UF/DF Omega™ Membrane Cassette
Fluorodyne® II DFL / Fluorodyne EX EDF
Sterilising Filtration
Formulation and Fill
*Proposed process based on data collected during this study
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