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: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: M
authors.initials: M
authors.lastname: Vargas
authors.forename: Á
authors.initials: Á
authors.lastname: Segura
authors.forename: Y-W
authors.initials: YW
authors.lastname: Wu
authors.forename: M
authors.initials: M
authors.lastname: Herrera
authors.forename: M-L
authors.initials: ML
authors.lastname: Chou
authors.forename: M
authors.initials: M
authors.lastname: Villalta
authors.forename: G
authors.initials: G
authors.lastname: León
authors.forename: T
authors.initials: T
authors.lastname: Burnouf
mesh_terms: D002210:Caprylates / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D015956:Factor XIIa / Q000032:analysis; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry; D010949:Plasma / Q000737:chemistry*; D013917:Thrombin / Q000096:biosynthesis
chemical_list: D002210:Caprylates; D007074:Immunoglobulin G; D009842:Oligopeptides; C015608:benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; C018788:valyl-leucyl-lysine 4-nitroanilide; C026148:prolyl-phenylalanyl-arginine-4-nitroanilide; D015956:Factor XIIa; D013917:Thrombin; C031492:octanoic acid
keywords: aqueous two-phase system; chromatography; immunoglobulin; plasma fractionation; thrombogenicity