title: Factors affecting endotoxin removal from recombinant therapeutic proteins by anion exchange chromatography.
abstract: Removal of endotoxins from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of the bacterial expression systems widely used to manufacture therapeutic proteins. In this study we investigated various parameters affecting anion exchange chromatography to selectively remove endotoxins from therapeutic proteins. NY-ESO-1, Melan-A, and SSX-2 are different recombinant proteins used in this study, all of them are cancer antigens currently developed as potential immunotherapeutic agents. We found that by using a commercially available Q XL resin in a flow-through mode, endotoxin could be effectively removed from these proteins while maintaining very acceptable protein yields. The ratio of resin volume to endotoxin load was analyzed to determine the endotoxin binding capacity of the resin. In our hands at least 900,000 endotoxin units (EU) could be loaded per ml of Q XL resin. Solution conductivity could be increased to 20 mS/cm to minimize protein loss by weakening protein-resin attraction, and pH could be increased to enhance endotoxin removal by weakening endotoxin-protein attraction. Endotoxin levels were ultimately decreased to below 0.5 EU per microg of protein, an over 2000-fold reduction in this single step. A successful scale-up of these processes in which column volume was increased 100-fold was performed under cGMP conditions with over 80% protein recovery.
authors.affiliation: Graduate Field of Chemical Engineering, Cornell University, Ithaca, NY 14853, USA. rhc27@cornell.edu
authors.forename: Rishard H
authors.initials: RH
authors.lastname: Chen
authors.forename: Chung-Jr
authors.initials: CJ
authors.lastname: Huang
authors.forename: Brian S
authors.initials: BS
authors.lastname: Newton
authors.forename: Gerd
authors.initials: G
authors.lastname: Ritter
authors.forename: Lloyd J
authors.initials: LJ
authors.lastname: Old
authors.forename: Carl A
authors.initials: CA
authors.lastname: Batt
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004553:Electric Conductivity; D004731:Endotoxins / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D058965:MART-1 Antigen; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D004731:Endotoxins; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011506:Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C093962:synovial sarcoma X breakpoint proteins