title: Release of proteolytic activity following reduction in therapeutic human serum albumin containing products: detection with a new neoepitope endopeptidase immunoassay.
abstract: Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln(197) and Arg(198) under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg(198) and Ala(199) was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125-30 mg of HSA. An excellent correlation (R(2)=0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α(2)-antiplasmin-plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg-Ala cleaving activity.
authors.affiliation: National Institute for Biological Standards and Control, Health Protection Agency, Division of Bacteriology, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. russell.jones@nibsc.hpa.org.uk
authors.forename: Russell G A
authors.initials: RG
authors.lastname: Jones
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Liu
authors.forename: Chris
authors.initials: C
authors.lastname: Halls
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Thorpe
authors.forename: Colin
authors.initials: C
authors.lastname: Longstaff
authors.forename: Paul
authors.initials: P
authors.lastname: Matejtschuk
authors.forename: Dorothea
authors.initials: D
authors.lastname: Sesardic
mesh_terms: D000418:Albumins / Q000737:chemistry; D001905:Botulinum Toxins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009498:Neurotoxins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D012709:Serum Albumin / Q000032:analysis*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry; D020840:Tissue Kallikreins / Q000737:chemistry; D014148:Tranexamic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D000418:Albumins; D000939:Epitopes; D009498:Neurotoxins; D010455:Peptides; D012709:Serum Albumin; D050825:Synaptosomal-Associated Protein 25; C064866:PKSI 527; D010649:Phenylalanine; D014148:Tranexamic Acid; D010450:Endopeptidases; D020840:Tissue Kallikreins; D001905:Botulinum Toxins; D015032:Zinc; D003545:Cysteine