Human plasma-derived immunoglobulin G fractionated by an aqueous two-phase system, caprylic acid precipitation, and membrane chromatography has a high purity level and is free of detectable in vitro thrombogenic activity

M. Vargas, A. Segura, Y. W. Wu, M. Herrera, M. L. Chou, M. Villalta, G. León, T. Burnouf

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background and Objectives: Instituto Clodomiro Picado has developed an immunoglobulin G (IgG) plasma fractionation process combining a polyethylene glycol/phosphate aqueous two-phase system (ATPS), caprylic acid precipitation and anion-exchange membrane chromatography. We evaluated the purity and in vitro thrombogenicity of such IgG, in line with current international requirements. Materials and Methods: Contributions of the different production steps to reduce thrombogenicity were assessed at 0·2 l-scale, and then the methodology was scaled-up to a 10 l-scale and final products (n = 3) were analysed. Purity, immunoglobulin composition, and subclass distribution were determined by electrophoretic and immunochemical methods. The in vitro thrombogenic potential was determined by a thrombin generation assay (TGA) using a Technothrombin fluorogenic substrate. Prekallikrein activator (PKA), plasmin, factor Xa, thrombin and thrombin-like activities were assessed using S-2302, S-2251, S-2222, S-2238 and S-2288 chromogenic substrates, respectively, and FXI by an ELISA. Results: The thrombogenicity markers were reduced mostly during the ATPS step and were found to segregate mostly into the discarded liquid upper phase. The caprylic acid precipitation eliminated the residual procoagulant activity. The IgG preparations made from the 10 l-batches contained 100% gamma proteins, low residual IgA and undetectable IgM. The IgG subclass distribution was not substantially affected by the process. TGA and amidolytic activities revealed an undetectable in vitro thrombogenic risk and the absence of proteolytic enzymes in the final product. Conclusions: Fractionating human plasma by an ATPS combined with caprylic acid and membrane chromatography resulted in an IgG preparation of high purity and free of a detectable in vitro thrombogenic risk.

Original languageEnglish
Pages (from-to)169-177
Number of pages9
JournalVox Sanguinis
Volume108
Issue number2
DOIs
Publication statusPublished - Feb 1 2015

Fingerprint

Chromatography
Thrombin
Immunoglobulin G
Membranes
isoleucyl-prolyl-arginine-4-nitroanilide
prolyl-phenylalanyl-arginine-4-nitroanilide
benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide
S 2238
valyl-leucyl-lysine 4-nitroanilide
Factor XIIa
Factor Xa
Fibrinolysin
Fluorescent Dyes
Immunoglobulin A
Anions
Immunoglobulin M
Immunoglobulins
Peptide Hydrolases
Enzyme-Linked Immunosorbent Assay
Phosphates

Keywords

  • Aqueous two-phase system
  • Chromatography
  • Immunoglobulin
  • Plasma fractionation
  • Thrombogenicity

ASJC Scopus subject areas

  • Hematology
  • Medicine(all)

Cite this

Human plasma-derived immunoglobulin G fractionated by an aqueous two-phase system, caprylic acid precipitation, and membrane chromatography has a high purity level and is free of detectable in vitro thrombogenic activity. / Vargas, M.; Segura, A.; Wu, Y. W.; Herrera, M.; Chou, M. L.; Villalta, M.; León, G.; Burnouf, T.

In: Vox Sanguinis, Vol. 108, No. 2, 01.02.2015, p. 169-177.

Research output: Contribution to journalArticle

@article{21326117a41d42a483f994390154e4ff,
title = "Human plasma-derived immunoglobulin G fractionated by an aqueous two-phase system, caprylic acid precipitation, and membrane chromatography has a high purity level and is free of detectable in vitro thrombogenic activity",
abstract = "Background and Objectives: Instituto Clodomiro Picado has developed an immunoglobulin G (IgG) plasma fractionation process combining a polyethylene glycol/phosphate aqueous two-phase system (ATPS), caprylic acid precipitation and anion-exchange membrane chromatography. We evaluated the purity and in vitro thrombogenicity of such IgG, in line with current international requirements. Materials and Methods: Contributions of the different production steps to reduce thrombogenicity were assessed at 0·2 l-scale, and then the methodology was scaled-up to a 10 l-scale and final products (n = 3) were analysed. Purity, immunoglobulin composition, and subclass distribution were determined by electrophoretic and immunochemical methods. The in vitro thrombogenic potential was determined by a thrombin generation assay (TGA) using a Technothrombin fluorogenic substrate. Prekallikrein activator (PKA), plasmin, factor Xa, thrombin and thrombin-like activities were assessed using S-2302, S-2251, S-2222, S-2238 and S-2288 chromogenic substrates, respectively, and FXI by an ELISA. Results: The thrombogenicity markers were reduced mostly during the ATPS step and were found to segregate mostly into the discarded liquid upper phase. The caprylic acid precipitation eliminated the residual procoagulant activity. The IgG preparations made from the 10 l-batches contained 100{\%} gamma proteins, low residual IgA and undetectable IgM. The IgG subclass distribution was not substantially affected by the process. TGA and amidolytic activities revealed an undetectable in vitro thrombogenic risk and the absence of proteolytic enzymes in the final product. Conclusions: Fractionating human plasma by an ATPS combined with caprylic acid and membrane chromatography resulted in an IgG preparation of high purity and free of a detectable in vitro thrombogenic risk.",
keywords = "Aqueous two-phase system, Chromatography, Immunoglobulin, Plasma fractionation, Thrombogenicity",
author = "M. Vargas and A. Segura and Wu, {Y. W.} and M. Herrera and Chou, {M. L.} and M. Villalta and G. Le{\'o}n and T. Burnouf",
year = "2015",
month = "2",
day = "1",
doi = "10.1111/vox.12209",
language = "English",
volume = "108",
pages = "169--177",
journal = "Vox Sanguinis",
issn = "0042-9007",
publisher = "Blackwell Publishing Ltd",
number = "2",

}

TY - JOUR

T1 - Human plasma-derived immunoglobulin G fractionated by an aqueous two-phase system, caprylic acid precipitation, and membrane chromatography has a high purity level and is free of detectable in vitro thrombogenic activity

AU - Vargas, M.

AU - Segura, A.

AU - Wu, Y. W.

AU - Herrera, M.

AU - Chou, M. L.

AU - Villalta, M.

AU - León, G.

AU - Burnouf, T.

PY - 2015/2/1

Y1 - 2015/2/1

N2 - Background and Objectives: Instituto Clodomiro Picado has developed an immunoglobulin G (IgG) plasma fractionation process combining a polyethylene glycol/phosphate aqueous two-phase system (ATPS), caprylic acid precipitation and anion-exchange membrane chromatography. We evaluated the purity and in vitro thrombogenicity of such IgG, in line with current international requirements. Materials and Methods: Contributions of the different production steps to reduce thrombogenicity were assessed at 0·2 l-scale, and then the methodology was scaled-up to a 10 l-scale and final products (n = 3) were analysed. Purity, immunoglobulin composition, and subclass distribution were determined by electrophoretic and immunochemical methods. The in vitro thrombogenic potential was determined by a thrombin generation assay (TGA) using a Technothrombin fluorogenic substrate. Prekallikrein activator (PKA), plasmin, factor Xa, thrombin and thrombin-like activities were assessed using S-2302, S-2251, S-2222, S-2238 and S-2288 chromogenic substrates, respectively, and FXI by an ELISA. Results: The thrombogenicity markers were reduced mostly during the ATPS step and were found to segregate mostly into the discarded liquid upper phase. The caprylic acid precipitation eliminated the residual procoagulant activity. The IgG preparations made from the 10 l-batches contained 100% gamma proteins, low residual IgA and undetectable IgM. The IgG subclass distribution was not substantially affected by the process. TGA and amidolytic activities revealed an undetectable in vitro thrombogenic risk and the absence of proteolytic enzymes in the final product. Conclusions: Fractionating human plasma by an ATPS combined with caprylic acid and membrane chromatography resulted in an IgG preparation of high purity and free of a detectable in vitro thrombogenic risk.

AB - Background and Objectives: Instituto Clodomiro Picado has developed an immunoglobulin G (IgG) plasma fractionation process combining a polyethylene glycol/phosphate aqueous two-phase system (ATPS), caprylic acid precipitation and anion-exchange membrane chromatography. We evaluated the purity and in vitro thrombogenicity of such IgG, in line with current international requirements. Materials and Methods: Contributions of the different production steps to reduce thrombogenicity were assessed at 0·2 l-scale, and then the methodology was scaled-up to a 10 l-scale and final products (n = 3) were analysed. Purity, immunoglobulin composition, and subclass distribution were determined by electrophoretic and immunochemical methods. The in vitro thrombogenic potential was determined by a thrombin generation assay (TGA) using a Technothrombin fluorogenic substrate. Prekallikrein activator (PKA), plasmin, factor Xa, thrombin and thrombin-like activities were assessed using S-2302, S-2251, S-2222, S-2238 and S-2288 chromogenic substrates, respectively, and FXI by an ELISA. Results: The thrombogenicity markers were reduced mostly during the ATPS step and were found to segregate mostly into the discarded liquid upper phase. The caprylic acid precipitation eliminated the residual procoagulant activity. The IgG preparations made from the 10 l-batches contained 100% gamma proteins, low residual IgA and undetectable IgM. The IgG subclass distribution was not substantially affected by the process. TGA and amidolytic activities revealed an undetectable in vitro thrombogenic risk and the absence of proteolytic enzymes in the final product. Conclusions: Fractionating human plasma by an ATPS combined with caprylic acid and membrane chromatography resulted in an IgG preparation of high purity and free of a detectable in vitro thrombogenic risk.

KW - Aqueous two-phase system

KW - Chromatography

KW - Immunoglobulin

KW - Plasma fractionation

KW - Thrombogenicity

UR - http://www.scopus.com/inward/record.url?scp=84921310899&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84921310899&partnerID=8YFLogxK

U2 - 10.1111/vox.12209

DO - 10.1111/vox.12209

M3 - Article

C2 - 25469648

AN - SCOPUS:84921310899

VL - 108

SP - 169

EP - 177

JO - Vox Sanguinis

JF - Vox Sanguinis

SN - 0042-9007

IS - 2

ER -