title: Use of ferric thiocyanate derivatization for quantification of polysorbate 80 in high concentration protein formulations.
abstract: Quantitation of polysorbate 80 in high protein formulation using solid-phase extraction (SPE) followed by derivatization with cobalt thiocyanate and UV measurement of the complex at 620 nm resulted in lower recovery of polysorbate 80. Dilution of protein samples with water improved the recovery of polysorbate, however, it compromised the sensitivity of the method when cobalt thiocyanate was used for derivatization. The presented work discusses an evaluation of alternative approaches for increasing the sensitivity of the quantitation method for polysorbate using ferric thiocyanate and molybdenum thiocyanate. Ferric thiocyanate complex of polysorbate 80 exhibited the highest sensitivity among the metals thiocyanate evaluated in the current work. The improvement in the sensitivity through derivatization with ferric thiocyanate allowed 10-fold dilution of a 140 mg mL(-1) protein sample without affecting the recovery or compromising the sensitivity of polysorbate 80 quantitation, indicating that this methodology could be used as an alternate approach for the quantitation of polysorbate 80 in high concentration protein formulations. Stability of ferric thiocynate and cobalt thiocyanate complex was also studied. When these complexes were allowed to equilibrate for 1h between an organic layer containing polysorbate/thiocynate complex and an aqueous layer containing un-reacted metal thiocyanate, it resulted in the most reproducible UV absorbance measurements. The SPE method for quantification of polysorbate 80 using ferric thiocyanate for derivatization provided accuracy (% spike recovery) within 107%, reproducibility (%relative standard deviation) less than 11.7%. The method is linear from 0.0001 to 0.008% polysorbate 80 concentrations in the formulations with protein formulations as high as 140 mg mL(-1).
authors.affiliation: Process and Product Development, Amgen, Inc. One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Nimesh
authors.initials: N
authors.lastname: Savjani
authors.affiliation: Process and Product Development, Amgen, Inc. One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Eugene
authors.initials: E
authors.lastname: Babcock
authors.affiliation: Process and Product Development, Amgen, Inc. One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Hui Koon
authors.initials: HK
authors.lastname: Khor
authors.affiliation: Process and Product Development, Amgen, Inc. One Amgen Center Drive, Thousand Oaks, CA 91320, USA. Electronic address: araghani@amgen.com.
authors.forename: Anil
authors.initials: A
authors.lastname: Raghani
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007501:Iron / Q000737:chemistry*; D011136:Polysorbates / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D052616:Solid Phase Extraction / Q000379:methods*; D013861:Thiocyanates / Q000737:chemistry*
chemical_list: D011136:Polysorbates; D011506:Proteins; D013861:Thiocyanates; C103724:ferric thiocyanate; D007501:Iron
keywords: Derivatization; Ferric-thiocyanate; Polysorbate 80; Protein formulation; Quantitation; SPE