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1.
Talanta ; 130: 542-6, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25159444

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).


Subject(s)
Chromatography, High Pressure Liquid/methods , Iron/chemistry , Polysorbates/analysis , Proteins/analysis , Solid Phase Extraction/methods , Thiocyanates/chemistry
2.
Anal Biochem ; 433(2): 137-49, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23085118

ABSTRACT

Stable isotope labeling combined with mass spectrometry has been widely used in a diverse set of applications in the biochemistry and biomedical fields. When stable isotope-labeled proteins are produced via metabolic labeling of cell culture, a comprehensive assessment of the labeling pattern is imperative. In this study, we present a set of mass spectrometry-based bioanalytical tools developed for quantitatively tracing the levels of the stable isotopes incorporated into the recombinant proteins (monoclonal antibodies and Fc fusion proteins expressed in different host systems) that include total mass analysis, peptide mapping analysis, and amino acid analysis. We show that these three mass spectrometry-based analytical methods have distinctive advantages and limitations and that they are mutually complementary in evaluating the quality of stable isotope-labeled proteins. In addition, we show that the analytical techniques developed here are powerful tools to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Immunoglobulin Fc Fragments/biosynthesis , Isotope Labeling/methods , Recombinant Fusion Proteins/biosynthesis , Animals , CHO Cells , Cell Culture Techniques/methods , Cricetinae , Cricetulus , Mass Spectrometry/methods
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