title: Endotoxin deactivation by transient acidification.
abstract: Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide endotoxin found in the outer bacterial membrane. Endotoxin could interfere with in vitro biological assays and is the major pathological factor, which must be removed or inactivated before in vivo administration. Here we describe a one-step protocol in which the endotoxin activity on recombinant proteins is remarkably reduced by transient exposure to acidic conditions. Maximum endotoxin deactivation occurs at acidic pH below their respective isoelectric point (pI). This method does not require additional protein purification or separation of the protein from the endotoxin fraction. The endotoxin level was measured both in vitro and in vivo. For in vitro assessment we have utilized Limulus Amebocyte Lysate method for in vivo the pyrogenic test. We have tested the above-mentioned method with five different recombinant proteins, including a monoclonal antibody clone 5c8 against CD154 produced by hybridomas. More than 99% of endotoxin was deactivated in all of the proteins; the recovery of the protein after deactivation varied between maximum 72.9% and minimum 46.8%. The anti-CD154 clone 5c8 activity remained unchanged as verified by the measurement of binding capability to activated lymphocytes. Furthermore, the effectiveness of this method was not significantly altered by urea, commonly used in protein purification. This procedure provides a simple and cost-efficient way to reduce the endotoxin activity in antibodies and recombinant proteins.
authors.affiliation: Diabetes Research Institute, University of Miami Leonard Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Melina M
authors.initials: MM
authors.lastname: Ribeiro
authors.forename: Xiumin
authors.initials: X
authors.lastname: Xu
authors.forename: Dagmar
authors.initials: D
authors.lastname: Klein
authors.forename: Norma S
authors.initials: NS
authors.lastname: Kenyon
authors.forename: Camillo
authors.initials: C
authors.lastname: Ricordi
authors.forename: Maria Sueli S
authors.initials: MS
authors.lastname: Felipe
authors.forename: Ricardo L
authors.initials: RL
authors.lastname: Pastori
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004731:Endotoxins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D014508:Urea / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D004731:Endotoxins; D011994:Recombinant Proteins; D014508:Urea