title: Preparation of crotaline F-ab antivenom (CroFab) with automated mixing methods: in vitro observations.
abstract: BACKGROUND
  Crotaline Polyvalent Ovine Fab antivenom (CroFab, Savage Laboratories and Protherics Inc., Brentwood, TN, USA) preparation requires that the lyophilized powder be manually reconstituted before use. We compared automated methods for driving the product into solution with the standard manual method of reconstitution, and the effect of repeated rinsing of the product vial, on the per-vial availability of antivenom.
  
  
  METHODS
  Normal saline (NS, 10 mL) was added to 12 vials of expired CroFab. Vials were assigned in pairs to each of six mixing methods, including one pair mixed manually as recommended by the product package insert. Each vial's contents were diluted to a final volume of 75 mL of normal saline. Protein concentration was measured with a colorimetric assay. The fluid left in each vial was removed and the vial was washed with 10 mL NS.
  
  
  RESULTS
  Total protein yield from each step was calculated. There was no significant change in protein yield among three of five automated mixing methods when compared to manual reconstitution. Repeat rinsing of the product vial with an additional 10 mLs of fluid added to the protein yield regardless of the mixing method used.
  
  
  CONCLUSIONS
  We found slightly higher protein yields with all automated methods compared to manual mixing, but only two of five comparisons with the standard mixing method demonstrated statistical significance. However, for all methods tested, the addition of a second rinsing and recovery step increased the amount of protein recovered considerably, presumably by allowing solution of protein trapped in the foamy residues.
  
  
  CONCLUSIONS
  Automated mixing methods and repeat rinsing of the product vial may allow higher protein yields in the preparation of CroFab antivenom.
authors.affiliation: Emergency Medicine, Olive View Medical Center, David Geffen School of Medicine at UCLA, Sylmar, CA, USA. raisvohra@hotmail.com
authors.forename: Rais
authors.initials: R
authors.lastname: Vohra
authors.forename: Michael
authors.initials: M
authors.lastname: Kelner
authors.forename: Richard F
authors.initials: RF
authors.lastname: Clark
mesh_terms: D000997:Antivenins / Q000737:chemistry*; D001331:Automation; D004339:Drug Compounding / Q000379:methods*; D004348:Drug Labeling; D004355:Drug Stability; D004356:Drug Storage; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008962:Models, Theoretical; D012996:Solutions
chemical_list: D000997:Antivenins; C431183:Crotalidae Polyvalent immune Fab; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D012996:Solutions