title: Recombinant human granulocyte colony stimulating factor pre-screening and screening of stabilizing carbohydrates and polyols.
abstract: 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.
authors.affiliation: Pliva Croatia Ltd, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia. renata.pavisic@pliva.com
authors.forename: Renata
authors.initials: R
authors.lastname: Pavisić
authors.forename: Karlo
authors.initials: K
authors.lastname: Hock
authors.forename: Ivana
authors.initials: I
authors.lastname: Mijić
authors.forename: Anita
authors.initials: A
authors.lastname: Horvatić
authors.forename: Martina
authors.initials: M
authors.lastname: Gecan
authors.forename: Mirela
authors.initials: M
authors.lastname: Sedić
authors.forename: Mirjana Bukvić
authors.initials: MB
authors.lastname: Krajacić
authors.forename: Mario
authors.initials: M
authors.lastname: Cindrić
mesh_terms: D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011108:Polymers / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012997:Solvents / Q000737:chemistry; D013678:Technology, Pharmaceutical / Q000379:methods; D013997:Time Factors
chemical_list: D002241:Carbohydrates; D005079:Excipients; D011108:Polymers; D011994:Recombinant Proteins; D012997:Solvents; C024617:polyol; D016179:Granulocyte Colony-Stimulating Factor