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  • 11
    Digitale Medien
    Digitale Medien
    Stability of Human Serum Albumin During Bioprocessing: Denaturation and Aggregation During Processing of Albumin Paste (2000)
    Springer
    Pharmaceutical research 17 (2000), S. 391-396 
    Details
    ISSN: 1573-904X
    Schlagwort(e): albumin ; human serum ; aggregation ; bioprocessing ; plasma proteins ; infrared spectroscopy
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract Purpose. To assess the impact of various bioprocessing steps on thestability of freshly precipitated human serum albumin (HSA) obtainedfrom pooled human plasma.Methods. After initial precipitation of HSA from plasma, the resultantpaste is either (a) lyophilized or (b) washed with acetone and thenair-dried in order to obtain a dry powder. The structure of HSA wasexamined using Fourier transform infrared (IR) spectroscopy. Theextent of aggregation of redissolved HSA was measured using bothdynamic light scattering and SDS-polyacrylamide gel electrophoresis(SDS-PAGE).Results. Both lyophilization and air-drying perturb the secondarystructural composition of HSA, as detected by infrared (IR) spectroscopy.Upon dissolution of dried paste, most of the protein refolds to anative-like conformation. However, a small fraction of the protein moleculesform soluble aggregates that can be detected by both dynamic lightscattering and SDS-PAGE. The level of aggregation is so low that itcould not be detected in the bulk by either circular dichroism orIR spectroscopy. The lyophilized protein, which appears to be moreunfolded in the solid state than the acetone washed/air-dried material,exhibits a higher level of aggregation upon dissolution.Conclusions. There is a direct correlation between the extent ofunfolding in the solid state and the amount of soluble aggregate presentafter dissolution. Moreover, the presence of the aggregates persiststhroughout the remainder of the purification process, which includesdissolution, chromatography, sterile filtration and viral inactivationsteps. Analytical methods used to monitor the stability ofbiopharmaceuticals in the final product can be used to assess damage inflictedduring processing of protein pharmaceuticals.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1007564601210
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  • 12
    Digitale Medien
    Digitale Medien
    Hydrophobic Ion Pairing: Altering the Solubility Properties of Biomolecules (1998)
    Springer
    Pharmaceutical research 15 (1998), S. 188-193 
    Details
    ISSN: 1573-904X
    Schlagwort(e): ion pairing ; ionic detergents ; solubility ; controlled release ; proteins
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract The high aqueous solubility of ionic compounds can be attributed to the ease of solvation of the counter ions. Replacement of the counter ions with ionic detergents dramatically alters the solubility properties of the molecule. Not only does the aqueous solubility drop precipitously, but the solubility in organic phases increases as well. Consequently, the partition coefficient changes by orders of magnitude. This ion pairing phenomenon, which we term hydrophobic ion pairing (HIP), has been extended to polyelectrolytes, such as proteins and polynucleotides. These materials form HIP complexes that dissolve in a range of organic solvents, often with retention of native structure and enzymatic activity. The HIP process has been used to purify protein mixtures, conduct enzymatic reactions in nonaqueous environments, increase structural stability, enhance bioavailability, and prepare new dosage forms.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1011998014474
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  • 13
    Digitale Medien
    Digitale Medien
    Stability of the Thrombolytic Protein Fibrolase: Effect of Temperature and pH on Activity and Conformation (1991)
    Springer
    Pharmaceutical research 8 (1991), S. 1103-1112 
    Details
    ISSN: 1573-904X
    Schlagwort(e): circular dichroism ; metalloprotein ; protein activity ; protein stability ; protein structure ; thrombolytic
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (∼22°C). At 37°C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alphahelical structure was lost in a cooperative transition (T m of 50°C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the T m determined by NMR was 46°C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43°C at pH 5. Protein concentrations determined over the pH range 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1015842032164
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  • 14
    Digitale Medien
    Digitale Medien
    Selective Precipitation of Interleukin-4 Using Hydrophobic Ion Pairing: A Method for Improved Analysis of Proteins Formulated with Large Excesses of Human Serum Albumin (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 1492-1495 
    Details
    ISSN: 1573-904X
    Schlagwort(e): hydrophobic ion pairing ; interleukin-4 ; protein analysis ; HPLC ; human serum albumin ; formulation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract In order to ensure the stability of protein pharmaceuticals, human serum albumin (HSA) is often added as an excipient, frequently in large excess. This makes chromatographic analysis of the stability of the active protein difficult. In the case of interleukin-4 (IL-4), separation from HSA can be achieved to some degree by size exclusion chromatography, but some HSA co-elutes with the IL-4. Hydrophobic ion pairing provides a method for selective precipitation of IL-4 from HSA. Hydrophobic ion pairing involves the electrostatic interaction of ionic detergents with oppositely charged polypeptides. Even when HSA is present in fifty-fold excess (w/w), the resulting precipitate contains greater than 70% of the IL-4. Selective precipitation with SDS produces enhancements in IL-4 over HSA of more than 2000-fold. This approach permits subsequent facile analysis of IL-4 by conventional reverse phase HPLC.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1018916627891
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  • 15
    Digitale Medien
    Digitale Medien
    Thermal Stability of Low Molecular Weight Urokinase During Heat Treatment. II. Effect of Polymeric Additives (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 1004-1008 
    Details
    ISSN: 1573-904X
    Schlagwort(e): protein stability ; aggregation ; turbidimetry ; urokinase ; formulation ; additives, polymeric
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract Turbidimetric or light scattering assays can be used to determine the extent of aggregation in protein formulations. Using low molecular weight urokinase (LMW-UK) as a model protein, the effect of polymeric additives on heat-induced aggregation was evaluated. Previous work has shown that under 60°C heat treatment, LMW-UK initially denatures and the unfolded protein associates to form soluble aggregates. Eventually, these aggregates associate to form a precipitate. The effects of polymers on the initial aggregation phase was examined. Hydroxyethyl (heta) starch, polyethylene glycol 4000, and gelatin were found to be effective, concentration-dependent inhibitors of aggregation, whereas polyvinylpyrrolidone (PVP) and polyethylene glycol 300 were ineffective. Overall, the effect of polymeric additives on the stability of thermally-stressed LMW-UK can be accounted for by preferential exclusion of the solute from the surface of the protein.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1018935420680
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  • 16
    Digitale Medien
    Digitale Medien
    Stability of Protein Pharmaceuticals (1989)
    Springer
    Pharmaceutical research 6 (1989), S. 903-918 
    Details
    ISSN: 1573-904X
    Schlagwort(e): protein stability ; biotechnology ; mutagenesis ; denaturation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications. Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development. Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties. These properties pose difficult stability problems. A summary of both chemical and physical decomposition pathways for proteins is given. Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination. Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces. Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1015929109894
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  • 17
    Digitale Medien
    Digitale Medien
    Effect of Zinc Binding on the Structure and Stability of Fibrolase, a Fibrinolytic Protein from Snake Venom (1992)
    Springer
    Pharmaceutical research 9 (1992), S. 870-877 
    Details
    ISSN: 1573-904X
    Schlagwort(e): fibrolase ; metalloprotease ; zinc binding ; enzyme ; sequence ; protein structure
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie
    Notizen: Abstract Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an α-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetet-raacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an α-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1023/A:1015840613799
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  • 18
    Digitale Medien
    Digitale Medien
    Approaches for increasing the solution stability of proteins (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 506-512 
    Details
    ISSN: 0006-3592
    Schlagwort(e): protein stabilization ; urokinase ; denaturation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. © 1995 John Wiley & Sons, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1002/bit.260480513
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  • 19
    Digitale Medien
    Digitale Medien
    Enhanced solubility of proteins and peptides in nonpolar solvents through hydrophobic ion pairing (1993)
    New York : Wiley-Blackwell
    Biopolymers 33 (1993), S. 927-932 
    Details
    ISSN: 0006-3525
    Schlagwort(e): Chemistry ; Polymer and Materials Science
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Chemie und Pharmazie
    Notizen: A new technique for enhancing the solubility of peptides and proteins in organic solvents is described. Complexation of polypeptides with stoichiometric amounts of an anionic detergent, such as sodium dodecyl sulfate (SDS), produces diminished aqueous solubility, but enhanced solubility in organic solvents. Consequently, the partitioning of a polypeptide into a nonpolar solvent can be increased by two to four orders of magnitude. In the case of an insulin-SDS complex, the solubility in 1-octanol is more than tenfold greater than in water. In 1-octanol, the native structure of insulin remains intact, as determined by CD spectroscopy, and the thermal denaturation temperature (Tm) is increased by approximately 50°C relative to unfolding in water. Finally, peptides and proteins can be extracted back into an aqueous phase provided the chloride concentration is sufficient to displace bound detergent molecules. © 1993 John Wiley & Sons, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1002/bip.360330608
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  • 20
    Digitale Medien
    Digitale Medien
    Molecular orbital calculations on the oligopeptides netropsin, distamycin and related compounds (1986)
    New York : Wiley-Blackwell
    Biopolymers 25 (1986), S. 2065-2082 
    Details
    ISSN: 0006-3525
    Schlagwort(e): Chemistry ; Polymer and Materials Science
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Chemie und Pharmazie
    Notizen: The electronic structure of the antibiotics netropsin, distamycin A, and related compounds has been examined by various theoretical models. Calculations of both the Pariser-Parr-Pople (PPP) and the semiempirical CNDO/S types can account for the absorption spectrum of netropsin and distamycin A. The CD spectrum has been calculated for the conformation of netropsin found in the crystal structure of a netropsin/DNA dodecamer. Not all the CD spectral features can be attributed entirely to the chiral conformation of netropsin, indicating that there are significant interactions between netropsin and DNA. A CD calculation was also performed for distamycin A in a similar conformation. An examination of the charge-density maps of the excited states suggests that there is substantial charge transfer from the pyrrole ring to either of the adjacent peptide linkages in these systems. At higher energies, even longer distance charge transfer can be observed. Similar behavior was seen in the monomers pyrrole-2-carboxamide and 3-(formylamino)pyrrole.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
    URL: http://dx.doi.org/10.1002/bip.360251104
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