ABSTRACT
Yeastolates, triton X-100 (TX-100) and methotrexate (MTX) are common process-related impurities (PRI) in cell-based bioproduction of many active biopharmaceuticals. In this study, a reverse phase high performance liquid chromatography (RP-HPLC) method coupled with ultraviolet (UV) detection was developed for simultaneous determination and quantitation of these impurities. The chromatographic separation was achieved using a Jupiter C4 column and analyses of yeastolates, TX-100 and MTX were monitored at 257, 280 and 302 nm, respectively. The method was further validated with respect to selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The limits of quantitation for yeastolates, TX-100 and MTX were determined to be 27 ppm, 10 ppm and 41 ppb, respectively. Finally, the suitability of the method for analyses of recombinant human hyaluronidase (rHuPH20) in-process (viral inactivation, QFF, PS, APB and CHT filtered, final viral filtrate) and final manufacturing materials was demonstrated, and trace levels of yeastolates, TX-100 and MTX were reliably measured except for three matrices early in the purification process in which TX-100 was not accurately determined due to interfering effects.
Subject(s)
Chromatography, Reverse-Phase/methods , Culture Media/analysis , Methotrexate/analysis , Octoxynol/analysis , Cell Adhesion Molecules/isolation & purification , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase/standards , Culture Media/chemistry , Drug Contamination , Humans , Hyaluronoglucosaminidase/isolation & purification , Recombinant Proteins/isolation & purification , Reproducibility of Results , Sensitivity and Specificity , YeastsABSTRACT
Protein-protein interactions between SHEP and Cas proteins influence cellular signaling through tyrosine kinases, as well as integrin-mediated signaling, and may be linked to antiestrogen resistance. Data from past studies suggests that association between SHEP and Cas proteins is critical for these cellular effects. In this study, the interacting domains of each protein were co-expressed in bacteria and a soluble stable complex was purified. Deuterium exchange mass spectrometry was used to define regions that are buried when SHEP1 is in complex with Cas. The results reveal four segments in SHEP1 that are highly protected, including a region (residues 619-640) that contains a key residue, tyrosine 635, required for association with Cas. This region is predominately hydrophilic, yet remains protected from solvent in the complex.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Crk-Associated Substrate Protein/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Crk-Associated Substrate Protein/metabolism , Deuterium , Humans , Mice , Protein Binding , Protein Structure, Tertiary , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Solvents/chemistryABSTRACT
RIZ1 (PRDM2) and PRDI-BF1 (PRDM1) are involved in B cell differentiation and the development of B cell lymphomas. These proteins are expressed in two forms that differ by the presence or absence of a PR domain. The protein product that retains the PR domain is anti-tumorigenic while the product that lacks the PR domain is oncogenic and over-expressed in tumor cells. The conserved PR domain is homologous to the SET domain from a family of histone methyltransferases. RIZ1 is also a histone methyltransferase and methylates lysine 9 in histone H3. This activity has been mapped to the PR domain. In the present study, deuterium exchange mass spectrometry was used to define the structural boundaries of the RIZ1 PR domain and to map sites of missense mutations that occur in human cancers and reduce methyltransferase activity. Flexible segments were selectively deleted to produce protein products that crystallize for structural studies. Segments at the carboxyl terminus of the PR domain that are involved in methylation of H3 were shown to be flexible, similar to SET domains, suggesting that the PR and SET methyltransferases may belong to an emerging class of proteins that contain mobile functional regions.
Subject(s)
DNA-Binding Proteins/chemistry , Nuclear Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Base Sequence , Crystallization , DNA Primers , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Electrophoresis, Polyacrylamide Gel , Histone-Lysine N-Methyltransferase , Humans , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity , Transcription Factors/metabolism , Transcription Factors/physiologyABSTRACT
The yeast Saccharomyces cerevisiae F1F0-ATPase epsilon-subunit (61 residues) was synthesized by the solid-phase peptide approach under both acidic and basic strategies. Only the latter strategy allowed us to obtain a pure epsilon-subunit. The strong propensity of the protein to produce few soluble dimeric species depending on pH has been proved by size-exclusion chromatography, electrophoresis and mass spectrometry. A circular dichroism study showed that an aqueous solution containing 30% trifluoroethanol or 200 mM sodium dodecyl sulphate is required for helical folding. In both solvents at acidic pH, the epsilon-subunit is soluble and monomeric.
