Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability.

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).

[1]  Kinam Park,et al.  Controlled drug delivery : designing technologies for the future , 2000 .

[2]  D. Brooks,et al.  A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol. , 1986, Analytical biochemistry.

[3]  K. Griebenow,et al.  Structure of poly(ethylene glycol)-modified horseradish peroxidase in organic solvents: infrared amide I spectral changes upon protein dehydration are largely caused by protein structural changes and not by water removal per se. , 2002, Biophysical journal.

[4]  E. Schacht,et al.  Review: Poly(Ethylene Glycol)s with Reactive Endgroups. II. Practical Consideration for the Preparation of Protein-PEG Conjugates , 1996 .

[5]  S. Nakamura,et al.  Multiple forms of glucose oxidase with different carbohydrate compositions. , 1981, Biochimica et biophysica acta.

[6]  A. Moosmann,et al.  Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media. , 2010, Journal of chromatography. A.

[7]  Lizhong He,et al.  Process development in affinity separation of glycoconjugates with lectins as ligands. , 2003, Journal of chromatography. A.

[8]  T. Ueno,et al.  Polyethylene glycol-modified concanavalin A as an effective agent to stimulate anti-tumor cytotoxicity. , 2000, Cancer detection and prevention.

[9]  H. Chase,et al.  Prediction of the performance of preparative affinity chromatography. , 1984, Journal of chromatography.

[10]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[11]  Palle Schneider,et al.  Directed evolution of a fungal peroxidase , 1999, Nature Biotechnology.

[12]  C. Anfinsen,et al.  [31] Affinity chromatography , 1971 .

[13]  J. Lee,et al.  Stability of PEGylated salmon calcitonin in nasal mucosa. , 2004, Journal of pharmaceutical sciences.

[14]  G. Hermanson,et al.  Immobilized Affinity Ligand Techniques , 1992 .

[15]  Y. Tsutsumi,et al.  Molecular design of hybrid tumour necrosis factor alpha with polyethylene glycol increases its anti-tumour potency. , 1995, British Journal of Cancer.

[16]  M. Ramjeesingh,et al.  Stabilization of biologically active proteins , 1989 .

[17]  M. Rito‐Palomares,et al.  Separation of PEGylated from unmodified ribonuclease A using sepharose media , 2009 .

[18]  H. Chase,et al.  Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon. , 2003, Biotechnology and bioengineering.

[19]  P. Caliceti,et al.  Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine). , 2000, Farmaco.

[20]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[21]  J. Kopeček,et al.  Enzymatic activity of chymotrypsin and its poly(ethylene glycol) conjugates toward low and high molecular weight substrates. , 1993, Bioconjugate chemistry.

[22]  S. W. Kim,et al.  Glucose-induced release of glycosylpoly(ethylene glycol) insulin bound to a soluble conjugate of concanavalin A. , 1997, Bioconjugate chemistry.

[23]  M. Mccaman,et al.  Precise and comparative pegylation analysis by microfluidics and mass spectrometry. , 2006, Analytical biochemistry.

[24]  C. Ó’Fágáin.  Enzyme stabilization: recent experimental progress , 2003 .

[25]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[26]  J. Marty,et al.  Kinetics of thermal inactivation of horseradish peroxidase : stabilizing effect of methoxypoly(ethylene glycol) , 1998 .

[27]  G. Saab-Rincón,et al.  High temperature biocatalysis by chemically modified cytochrome C. , 2002, Bioconjugate chemistry.

[28]  R. Hu,et al.  Bioactivities of ricin retained and its immunoreactivity to anti-ricin polyclonal antibodies alleviated through pegylation. , 2002, The international journal of biochemistry & cell biology.

[29]  Katta G. Murty,et al.  On KΔ , 1986, Discret. Appl. Math..

[30]  Sunanda R. Narayanan,et al.  Preparative affinity chromatography of proteins , 1990 .

[31]  D. F. Smith,et al.  Purification of Forssman and human blood group A glycolipids by affinity chromatography on immobilized Helix pomatia lectin. , 1988, Analytical biochemistry.

[32]  Solvation Energy and Thermal Stability of Hydrophilization-Modified α-Chymotrypsin , 1999, Journal of protein chemistry.

[33]  D. Combes,et al.  Thermostability of modified enzymes : a detailed study , 1999 .

[34]  Zhimin He,et al.  Kinetic study of thermal inactivation for native and methoxypolyethylene glycol modified trypsin , 2000 .

[35]  Dan Qin,et al.  A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example. , 2010, Analytical biochemistry.

[36]  B. Niemeyer,et al.  Evaluation of two different Concanavalin A affinity adsorbents for the adsorption of glucose oxidase. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[37]  R. Misra,et al.  Biomaterials , 2008 .

[38]  M. Uhlén,et al.  Stability towards alkaline conditions can be engineered into a protein ligand. , 2000, Journal of biotechnology.

[39]  Toshio Kajiuchi,et al.  Development of effective modified cellulase for cellulose hydrolysis process , 1995, Biotechnology and Bioengineering.

[40]  K. Griebenow,et al.  Effect of the covalent modification with poly(ethylene glycol) on alpha-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres. , 2005, Journal of pharmaceutical sciences.

[41]  A. Andrews,et al.  Glycosidases in organic solvents: II. Transgalactosylation catalysed by polyethylene glycol-modified β-galactosidase , 1990 .

[42]  Smith Df,et al.  Lectin affinity chromatography of glycolipids and glycolipid-derived oligosaccharides. , 1989 .

[43]  Ashok Gowda,et al.  Concanavalin A for in vivo glucose sensing: a biotoxicity review. , 2006, Biosensors & bioelectronics.

[44]  Kinam Park,et al.  Glucose-Binding Property of Pegylated Concanavalin a , 2001, Pharmaceutical Research.

[45]  A. Habeeb,et al.  Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. , 1966, Analytical biochemistry.

[46]  J. Liesienė,et al.  Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography. , 2006, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[47]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[48]  H. Sato,et al.  Enzymatic procedure for site-specific pegylation of proteins. , 2002, Advanced drug delivery reviews.

[49]  Bernd Niemeyer,et al.  Comparison of modified supports on the base of glycoprotein interaction studies and of adsorption investigations. , 2005, Journal of chromatography. A.

[50]  J. V. Van Alstine,et al.  Prediction of the viscosity radius and the size exclusion chromatography behavior of PEGylated proteins. , 2004, Bioconjugate chemistry.

[51]  T. Park,et al.  Stabilization of recombinant interferon-α by pegylation for encapsulation in PLGA microspheres , 2003 .

[52]  J. V. Alstine,et al.  PEG-proteins: Reaction engineering and separation issues , 2006 .