Recombinant human granulocyte colony stimulating factor pre-screening and screening of stabilizing carbohydrates and polyols.

Protein stabilization by solvent additives is frequently used concept in formulation development, although new technologies implemented over the past decade can improve protein biophysical as well as clinical properties by protein structural design (e.g. PEGylation, acylation, hesylation). The scope of this work was to evaluate the effect of chosen carbohydrate or polyol stabilizer in the formulation; firstly on linear peptide sequences on instable model of rHuG-CSF cleaved macromolecule by novel method named protein and peptide stabilizer pre-screening PPSP (formulated tryptic digest mixture stability evaluation in 54 h) and on overall stability of rHuG-CSF macromolecule by quantifying all relevant degradation parameters. Comprehensive protein stabilizing screening study included conformational analysis of formulated rHuG-CSF protein to obtain information on its secondary structure conformational stability. Protein aggregation induced by modulating conditions in solution (e.g. thermal stress and agitation) was monitored over discrete time periods. Oxidation and deamidation, as well as truncation or hydrolysis were accurately quantified. Together with pre-screening data, obtained by fast and resourceful amino acid sequence degradation analysis by LC-MS, statistical data evaluation of stabilizing contribution of substances selected from group of carbohydrates and polyols was performed. According to the statistical interpretation of obtained results the stabilizers were ranked in the following order: turanose, D-trehalose, lactitol, acetate buffer (non-stabilized sample), xylitol, cellobiitol, sorbitol, D-lyxose, leucrose, sorbitol without polysorbate, cellobiose.

Determination of dithiothreitol in complex protein mixtures by HPLC-MS.

Dithiothreitol (DTT) and other reducing agents are typically used in refolding processes of recombinant human proteins during their purification from inclusion bodies. Due to its toxicity, it is essential to monitor the clearance of DTT throughout the analytical flow from the refolding phase to the final formulated product. Here we report a direct, simple, and fast liquid chromatography method using UV and tandem mass spectrometry (MS/MS) detection for DTT evaluation in complex protein mixtures. In aqueous solution DTT exists as an equilibrium mixture of the oxidized and the reduced form (H(2)DTT --> DTT(ox)) and the quantitation tools should therefore be applicable to both forms in a single step or in multiple steps. Oxidation of DTT with aqueous copper(II) nitrate trihydrate solution was introduced to determine a single oxidized compound, i. e. DTT(ox). Proteins and other components of high molecular masses were separated from DTT(ox) by ultrafiltration. Consequently, efficient separation of the DTT(ox )from other flow-through mixture components (sugars, polymers, salts, protein stabilizers) was achieved on an Atlantis dC(18) column. After chromatographic separation, DTT(ox) was selectively identified by UV absorbance at 285 nm or by selected reaction monitoring, measuring signal transition between m/z 151 --> 105. The method was validated in terms of specificity, accuracy, precision, linearity, and limit of quantification and detection. A reversed-phase HPLC separation method with atmospheric pressure chemical ionization and MS/MS detection in negative ion mode is highlighted as a viable alternative to currently existing quantitation methods involving DTT derivatization and HPLC fluorescence detection. The described approach offers simple, straightforward, selective, and high-throughput DTT quantitation in protein mixtures.

Functional analysis of human S-adenosylhomocysteine hydrolase isoforms SAHH-2 and SAHH-3.

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of AdoHcy to adenosine and homocysteine. Increased levels of AdoHcy may play a role in the development of cardiovascular diseases and numerous other conditions associated with hyperhomocysteinemia. Several polymorphic isoforms named SAHH-1 to 4 may be resolved by horizontal starch gel electrophoresis from red blood cells. We have identified the genetic background of isoforms SAHH-2 and SAHH-3. SAHH-2 represents the previously described polymorphism in exon 2 of the AdoHcyase gene (112 C>T; p.R38W). Isoform SAHH-3 is based on a new polymorphism in exon 3 (377 G>A), leading to the conversion of glycine to arginine at amino-acid position 123. To shed light on the effects of these polymorphisms on the molecular and catalytic properties of AdoHcyase, we made recombinant wild-type and polymorphic R38W and G123R enzymes for a comparative analysis. The amino-acid exchanges did not bring about major changes to the catalytic rates of the recombinant proteins. However, circular dichroism analysis showed that both polymorphisms effect the thermal stability of the recombinant protein in vitro, reducing the unfolding temperature by approximately 2.6 degrees C (R38W) and 1.5 degrees C (G123R) compared to wild-type protein. In view of the altered thermal stability, and slightly decreased enzymatic activity of polymorphic proteins (< or =6%), one may consider the analyzed AdoHcyase isoforms as risk markers for diseases caused by irregular AdoHcyase metabolism.