Preparative Purification of Recombinant Proteins: Current Status and Future Trends

Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

[1]  P. Gray,et al.  Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology. , 2003, Protein expression and purification.

[2]  S E Builder,et al.  Use of multifactorial analysis to develop aqueous two-phase systems for isolation of non-native IGF-I. , 1995, Bioseparation.

[3]  M. Morbidelli,et al.  Model simulation and experimental verification of a cation-exchange IgG capture step in batch and continuous chromatography. , 2011, Journal of chromatography. A.

[4]  Sonochemistry , 1990, Science.

[5]  D. Daines,et al.  Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae , 2012, PloS one.

[6]  F. B. Anspach,et al.  Removal of endotoxins by affinity sorbents. , 1995, Journal of chromatography. A.

[7]  R. Raines,et al.  The S.Tag fusion system for protein purification. , 2000, Methods in enzymology.

[8]  S. Cramer,et al.  Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants. , 1998, Journal of biotechnology.

[9]  K. Terpe Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems , 2002, Applied Microbiology and Biotechnology.

[10]  D. C. Azevedo,et al.  Dye Ligand Epoxide Chitosan/Alginate: A Potential New Stationary Phase for Human IgG Purification , 2012 .

[11]  D. Winzor,et al.  Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin. , 2000, Journal of chromatography. B, Biomedical sciences and applications.

[12]  Daniel I. C. Wang,et al.  Separation of proteins and viruses using two-phase aqueous micellar systems. , 1998, Journal of chromatography. B, Biomedical sciences and applications.

[13]  D. Ejima,et al.  Solvent modulation of column chromatography. , 2008, Protein and peptide letters.

[14]  D. Rylatt,et al.  Preparative‐scale fractionation by isoelectric trapping under nondenaturing conditions: Separation of egg white protein isoforms on a modified Gradiflow unit , 2005, Electrophoresis.

[15]  R. Razavi,et al.  Recombinant Complement Receptor 2 Radiolabeled with [99mTc(CO)3]+ : A Potential New Radiopharmaceutical for Imaging Activated Complement , 2011, PloS one.

[16]  Dong-Qiang Lin,et al.  Evaluating antibody monomer separation from associated aggregates using mixed-mode chromatography. , 2013, Journal of chromatography. A.

[17]  A. Seidler Introduction of a histidine tail at the N-terminus of a secretory protein expressed in Escherichia coli. , 1994, Protein engineering.

[18]  A. Le Saux,et al.  Purification of histidine-tagged mitochondrial ADP/ATP carrier: influence of the conformational states of the C-terminal region. , 2000, Protein expression and purification.

[19]  R. Ulevitch,et al.  A new method for reduction of endotoxin contamination from protein solutions. , 1987, Journal of immunological methods.

[20]  L. J. Cummings,et al.  Protein chromatography on hydroxyapatite columns. , 2009, Methods in enzymology.

[21]  Kurt Brorson,et al.  Robustness of virus removal by protein A chromatography is independent of media lifetime. , 2008, Journal of chromatography. A.

[22]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[23]  Ana M Azevedo,et al.  Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies. , 2007, Journal of chromatography. A.

[24]  S. Cramer,et al.  Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography. , 1995, Biotechnology and bioengineering.

[25]  H. Ihara,et al.  Reduction of endotoxin contamination of various crude vaccine materials by gram-negative bacteria using aminated poly(gamma-methyl L-glutamate) spherical particles. , 1993, Biological & pharmaceutical bulletin.

[26]  C. Batt,et al.  Factors affecting endotoxin removal from recombinant therapeutic proteins by anion exchange chromatography. , 2009, Protein expression and purification.

[27]  E. C. Beuvery,et al.  Two-step purification of a murine monoclonal antibody intended for therapeutic application in man. Optimisation of purification conditions and scaling up. , 1989, Journal of immunological methods.

[28]  S. Cramer,et al.  Antibiotics as low-molecular-mass displacers in ion-exchange displacement chromatography , 1995 .

[29]  D. S. Hage,et al.  Affinity chromatography: a review of clinical applications. , 1999, Clinical chemistry.

[30]  M. N. Gupta,et al.  Three-phase partitioning for simultaneous renaturation and partial purification of Aspergillus niger xylanase. , 2004, Biochimica et biophysica acta.

[31]  E. Russell Ritenour,et al.  Ultrasound: Its Chemical, Physical, and Biological Effects , 1989 .

[32]  Carlos C. Tarazaga,et al.  Physical cleaning by means of electric field in the ultrafiltration of a biological solution , 2006 .

[33]  Richard J. Wakeman,et al.  Additional techniques to improve microfiltration , 2002 .

[34]  M. Cotten,et al.  Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus. , 1994, Gene therapy.

[35]  Jose C. Merchuk,et al.  Mathematical modelling and computer simulation of aqueous two-phase continuous protein extraction☆ , 1996 .

[36]  V. A. Stewart,et al.  Phase 1 randomized double-blind safety and immunogenicity trial of Plasmodium falciparum malaria merozoite surface protein FMP1 vaccine, adjuvanted with AS02A, in adults in western Kenya. , 2007, Vaccine.

[37]  C. Lowe,et al.  Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand. , 2000, Journal of chromatography. B, Biomedical sciences and applications.

[38]  Adalberto Pessoa,et al.  Methods of endotoxin removal from biological preparations: a review. , 2007, Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques.

[39]  M. Nyström,et al.  Fractionation of BSA and myoglobin with modified and unmodified ultrafiltration membranes. , 1995, Bioseparation.

[40]  Daisuke Ejima,et al.  MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution. , 2009, Protein expression and purification.

[41]  C. Zaror,et al.  Mathematical modelling and simulation of aqueous two-phase continuous protein extraction. , 1992, Bioseparation.

[42]  S. Cramer,et al.  Ion-exchange displacement chromatography of proteins , 1993 .

[43]  Ekta Mahajan,et al.  Improving affinity chromatography resin efficiency using semi-continuous chromatography. , 2012, Journal of chromatography. A.

[44]  K. Brorson,et al.  Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. , 2003, Journal of chromatography. A.

[45]  P. Strömberg,et al.  Endotoxin reduction in monoclonal antibody preparations using arginine. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[46]  A.J.J. Straathof,et al.  The Proportion of Downstream Costs in Fermentative Production Processes , 2011 .

[47]  D. Drolet,et al.  Aptamer affinity chromatography: combinatorial chemistry applied to protein purification. , 1999, Journal of chromatography. B, Biomedical sciences and applications.

[48]  J. E. Rasmussen,et al.  Small-molecule affinity ligands for protein purification: combined computational enrichment and automated in-line screening of an optically encoded library. , 2010, Angewandte Chemie.

[49]  Qiqing Zhang,et al.  Synthesis and endotoxin removal properties of a novel affinity sorbent with poly(1-vinylimidazole) as the ligand , 2011 .

[50]  Kurt Brorson,et al.  The clearance of viruses and transmissible spongiform encephalopathy agents from biologicals. , 2005, Current opinion in biotechnology.

[51]  R. Randall,et al.  Construction, purification and immunogenicity of antigen-antibody-LTB complexes. , 1996, Vaccine.

[52]  K. Christensen,et al.  Membrane chromatography: protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases. , 2010, Journal of chromatography. A.

[53]  Effect of polyhistidine-tagging site on the stability of recombinant alginate lyase from Streptomyces sp. ALG-5 , 2012, Journal of Pharmaceutical Investigation.

[54]  Cui,et al.  Lysozyme separation by hollow-fibre ultrafiltration. , 2000, Biochemical engineering journal.

[55]  Marie Tichá,et al.  Some potentialities and drawbacks of contemporary size-exclusion chromatography. , 2003, Journal of biochemical and biophysical methods.

[56]  A. Zydney,et al.  Effect of Membrane Charge on Flow and Protein Transport during Ultrafiltration , 2006, Biotechnology progress.

[57]  Mojgan Kavoosi,et al.  Inexpensive one-step purification of polypeptides expressed in Escherichia coli as fusions with the family 9 carbohydrate-binding module of xylanase 10A from T. maritima. , 2004, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[58]  Sulakshana Jain,et al.  Purification of recombinant green fluorescent protein by three-phase partitioning. , 2004, Journal of chromatography. A.

[59]  K. Büssow,et al.  An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography , 2003, BMC biotechnology.

[60]  Dong-Qiang Lin,et al.  Preparation and adsorption behavior of a cellulose-based, mixed-mode adsorbent with a benzylamine ligand for expanded bed applications , 2008 .

[61]  A. Skerra,et al.  The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. , 1993, Protein engineering.

[62]  Y. Clonis,et al.  Molecular modeling for the design of a biomimetic chimeric ligand. Application to the purification of bovine heart L-lactate dehydrogenase. , 1999, Biotechnology and bioengineering.

[63]  A. Ahmad,et al.  Automated electrophoretic membrane cleaning for dead-end microfiltration and ultrafiltration , 2002 .

[64]  J. Lebowitz,et al.  Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag. , 2006, Protein expression and purification.

[65]  L. Noël,et al.  Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope , 2004, Plant Molecular Biology.

[66]  J. Lenormand,et al.  Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity. , 2012, Biochemical and biophysical research communications.

[67]  Jie Chen,et al.  The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process. , 2010, Journal of chromatography. A.

[68]  D. Prazeres,et al.  Stability and stabilisation of penicillin acylase , 1999 .

[69]  F. Regnier,et al.  High-performance liquid chromatography of biopolymers. , 1983, Science.

[70]  M. Etzel,et al.  Evaluation of an ion‐exchange membrane for the purification of plasmid DNA , 2003, Biotechnology and applied biochemistry.

[71]  Kun-Hsien Tsai,et al.  Dengue virus inactivation by minipool TnBP/Triton X‐45 treatment of plasma and cryoprecipitate , 2013, Vox sanguinis.

[72]  A. Jungbauer,et al.  The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. , 2001, Journal of biochemical and biophysical methods.

[73]  B. Hambly,et al.  Direct cloning of polymerase chain reaction products into the pinpoint Xa1‐T vector protein expression system , 1998, Electrophoresis.

[74]  Andreas Seidel-Morgenstern,et al.  Recombinant protein purification using gradient assisted simulated moving bed hydrophobic interaction chromatography. Part II: process design and experimental validation. , 2011, Journal of chromatography. A.

[75]  K. Stubenrauch,et al.  Purification of a viral coat protein by an engineered polyionic sequence. , 2000, Journal of chromatography. B, Biomedical sciences and applications.

[76]  Ivo Safarik,et al.  Overview of Magnetic Separations Used in Biochemical and Biotechnological Applications , 1997 .

[77]  J. Haynes,et al.  Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine. , 2003, Molecular and biochemical parasitology.

[78]  M. Morbidelli,et al.  Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP) , 2010, Biotechnology and bioengineering.

[79]  Massimo Morbidelli,et al.  A semicontinuous 3-column countercurrent solvent gradient purification (MCSGP) process. , 2008, Biotechnology and bioengineering.

[80]  P. Nygren Affinity ligands from biological combinatorial libraries. , 2011, Methods of biochemical analysis.

[81]  R. Gentz,et al.  6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. , 1994, Methods in molecular biology.

[82]  D. Esposito,et al.  Enhanced soluble protein expression using two new fusion tags. , 2006, Protein expression and purification.

[83]  Hakim Lounici,et al.  Electric fields applied in the ultrafiltration process , 2001 .

[84]  R Kuriyel,et al.  High performance tangential flow filtration. , 1997, Biotechnology and bioengineering.

[85]  J. Hang,et al.  High performance DNA purification using a novel ion exchange matrix. , 2008, Journal of biomolecular techniques : JBT.

[86]  P A Marichal-Gallardo,et al.  State‐of‐the‐art in downstream processing of monoclonal antibodies: Process trends in design and validation , 2012, Biotechnology progress.

[87]  Jürgen Hubbuch,et al.  High throughput screening techniques in downstream processing: Preparation, characterization and optimization of aqueous two-phase systems , 2007 .

[88]  Joe X Zhou,et al.  PROCESS DEVELOPMENT FOR ROBUST REMOVAL OF AGGREGATES USING CATION EXCHANGE CHROMATOGRAPHY IN MONOCLONAL ANTIBODY PURIFICATION WITH IMPLEMENTATION OF QUALITY BY DESIGN , 2012, Preparative biochemistry & biotechnology.

[89]  V. Klyushnichenko,et al.  Methods of preparation of recombinant cytokine proteins. , 1998, Protein expression and purification.

[90]  F. Tjerneld,et al.  Partitioning and Characterization of Tyrosine‐Tagged Green Fluorescent Proteins in Aqueous Two‐Phase Systems , 2004, Biotechnology progress.

[91]  S. Ignatova,et al.  Scale-up of counter-current chromatography: demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale. , 2009, Journal of chromatography. A.

[92]  R. Meyer,et al.  Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis. , 2004, Protein expression and purification.

[93]  Miao-Fang Lin,et al.  Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents. , 2005, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[94]  R. Kurnik,et al.  Buffer exchange using size exclusion chromatography, countercurrent dialysis, and tangential flow filtration: Models, development, and industrial application , 1995, Biotechnology and bioengineering.

[95]  Massimo Morbidelli,et al.  A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process , 2007, Biotechnology and bioengineering.

[96]  Arvind Rajendran,et al.  Simulated moving bed chromatography for the separation of enantiomers. , 2009, Journal of chromatography. A.

[97]  M. Chern,et al.  Single-step protein purification by back flush in ion exchange chromatography. , 2009, Analytical biochemistry.

[98]  Gary Walsh,et al.  Biopharmaceutical benchmarks 2010 , 2010, Nature Biotechnology.

[99]  Zhanfeng Cui,et al.  Protein separation using ultrafiltration — an example of multi-scale complex systems , 2005 .

[100]  T. Peeples,et al.  Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems. , 2004, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[101]  S. Lute,et al.  Normal‐flow virus filtration: detection and assessment of the endpoint in bioprocessing , 2005, Biotechnology and applied biochemistry.

[102]  Steven M Cramer,et al.  Prediction of the effect of mobile-phase salt type on protein retention and selectivity in anion exchange systems. , 2003, Analytical chemistry.

[103]  A. Denizli,et al.  Purification of Alcohol Dehydrogenase from Saccharomyces cerevisiae Using Magnetic Dye-Ligand Affinity Nanostructures , 2013, Applied Biochemistry and Biotechnology.

[104]  E. Omidinia,et al.  Process integration for the recovery and purification of recombinant Pseudomonas fluorescens proline dehydrogenase using aqueous two-phase systems. , 2013, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[105]  R. Bayer,et al.  Recovery and purification process development for monoclonal antibody production , 2010, mAbs.

[106]  S. Cramer,et al.  Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers , 1995 .

[107]  M. Moo-young,et al.  Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC). , 2012, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[108]  Massimo Morbidelli,et al.  Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP) , 2008, Biotechnology and Bioengineering.

[109]  Oscar-Werner Reif,et al.  Densonucleosis virus purification by ion exchange membranes. , 2004, Biotechnology and bioengineering.

[110]  C. Kepka,et al.  Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system. , 2003, Journal of Biotechnology.

[111]  Richard J. Wakeman,et al.  Modelling, simulation and process design of the filter cycle , 1990 .

[112]  Y. Duman,et al.  Three-Phase Partitioning as a Rapid and Easy Method for the Purification and Recovery of Catalase from Sweet Potato Tubers (Solanum tuberosum) , 2013, Applied Biochemistry and Biotechnology.

[113]  R. Ghosh,et al.  Simultaneous removal of leached protein-A and aggregates from monoclonal antibody using hydrophobic interaction membrane chromatography , 2012 .

[114]  Z. Su,et al.  An automatic system for multidimensional integrated protein chromatography. , 2010, Journal of chromatography. A.

[115]  D. Ejima,et al.  Arginine as an effective additive in gel permeation chromatography. , 2005, Journal of chromatography. A.

[116]  Ralf Kuriyel,et al.  High-performance tangential flow filtration: a highly selective membrane separation process , 2002 .

[117]  D. Ejima,et al.  The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. , 2007, Protein expression and purification.

[118]  T. Tosa,et al.  Specific removal of endotoxin from protein solutions by immobilized histidine , 1990, Biotechnology and applied biochemistry.

[119]  J A Asenjo,et al.  Protein purification using chromatography: selection of type, modelling and optimization of operating conditions , 2009, Journal of molecular recognition : JMR.

[120]  B. L. Thomas,et al.  Adsorptive membrane chromatography for purification of plasmid DNA. , 2003, Journal of chromatography. A.

[121]  C. Kepka,et al.  Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system. , 2004, Journal of chromatography. A.

[122]  Steven M. Cramer,et al.  Characterization of non-linear adsorption properties of dextran-based polyelectrolyte displacers in ion-exchange systems , 1993 .

[123]  P. Hinckley,et al.  Ion exchange chromatography of proteins and clearance of aggregates. , 2009, Current pharmaceutical biotechnology.

[124]  T. Okuyama,et al.  Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G. , 2012, Hybridoma.

[125]  Teodoro Espinosa-Solares,et al.  Macroscopic mass and energy balance of a pilot plant anaerobic bioreactor operated under thermophilic conditions , 2006, Applied biochemistry and biotechnology.

[126]  R. Carbonell,et al.  Affinity purification of fibrinogen using a ligand from a peptide library. , 2002, Biotechnology and bioengineering.

[127]  A. Zydney,et al.  Protein fractionation using electrostatic interactions in membranel filtration. , 1995, Biotechnology and bioengineering.

[128]  B. Abu-Sharkh,et al.  Protein Partitioning in Aqueous Two‐Phase Systems Composed of a pH‐Responsive Copolymer and Poly(ethylene glycol) , 2008, Biotechnology progress.

[129]  J. Barnwell,et al.  The Clinical-Grade 42-Kilodalton Fragment of Merozoite Surface Protein 1 of Plasmodium falciparum Strain FVO Expressed in Escherichia coli Protects Aotus nancymai against Challenge with Homologous Erythrocytic-Stage Parasites , 2005, Infection and Immunity.

[130]  Ana M Azevedo,et al.  Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. , 2009, Trends in biotechnology.

[131]  C. Huang,et al.  Enhancing the separation of nano-sized particles in low-salt suspensions by electrically assisted cross-flow filtration , 2007 .

[132]  N. Thompson,et al.  An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody. , 2004, Protein expression and purification.

[133]  Gerhard Schembecker,et al.  Small scale, modular and continuous: A new approach in plant design , 2012 .

[134]  A. Doyen,et al.  Impact of pH on ultrafiltration membrane selectivity during electrodialysis with ultrafiltration membrane (EDUF) purification of soy peptides from a complex matrix , 2013 .

[135]  B. Mattiasson,et al.  Two-Phase Affinity Partitioning of Animal Cells: Implications of Multipoint Interactions , 2002 .

[136]  A. Mahn,et al.  Methods of calculating protein hydrophobicity and their application in developing correlations to predict hydrophobic interaction chromatography retention. , 2009, Journal of chromatography. A.

[137]  Development of an epitope tag for the gentle purification of proteins by immunoaffinity chromatography: application to epitope-tagged green fluorescent protein. , 2003, Analytical biochemistry.

[138]  S. Cramer,et al.  Cation‐Exchange Displacement Chromatography of Proteins with Protamine Displacers: Effect of Induced Salt Gradients , 1992, Biotechnology progress.

[139]  B. Sarkar,et al.  Electric field enhanced fractionation of protein mixture using ultrafiltration , 2009 .

[140]  Matthew T. Aspelund,et al.  Purification of recombinant plant-made proteins from corn extracts by ultrafiltration , 2010 .

[141]  T. Przybycien,et al.  Alternative bioseparation operations: life beyond packed-bed chromatography. , 2004, Current opinion in biotechnology.

[142]  G. Ehrlich,et al.  Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display. , 2001, Journal of biochemical and biophysical methods.

[143]  Anna Nilsson,et al.  Cutinase-peptide fusions in thermoseparating aqueous two-phase systems. Prediction of partitioning and enhanced tag efficiency by detergent addition. , 2002, Journal of chromatography. A.

[144]  W. Strohl Therapeutic Monoclonal Antibodies: Past, Present, and Future , 2009 .

[145]  Adalberto Pessoa,et al.  LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system , 2010, Biotechnology progress.

[146]  Y. Clonis,et al.  Biomimetic dyes as affinity chromatography tools in enzyme purification. , 2000, Journal of chromatography. A.

[147]  Daniel G Bracewell,et al.  Optimising the design and operation of semi-continuous affinity chromatography for clinical and commercial manufacture. , 2013, Journal of chromatography. A.

[148]  G. C. Sarti,et al.  Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification. , 2011, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[149]  C. Filipe,et al.  Cascade ultrafiltration systems—Integrated processes for purification and concentration of lysozyme , 2010 .

[150]  Kurt Brorson,et al.  Impact of multiple re-use of anion-exchange chromatography media on virus removal. , 2005, Journal of chromatography. A.

[151]  J. Falke,et al.  Purification of proteins using polyhistidine affinity tags. , 2000, Methods in enzymology.

[152]  C. Lowe,et al.  Design, synthesis, and application of a Protein A mimetic , 1998, Nature Biotechnology.

[153]  D. Schomburg,et al.  High level expression and single-step purification of hexahistidine-tagged L-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set. , 2005, Protein expression and purification.

[154]  D. Ejima,et al.  Arginine improves protein elution in hydrophobic interaction chromatography. The cases of human interleukin-6 and activin-A. , 2007, Journal of chromatography. A.

[155]  Yan Sun,et al.  Ligands for mixed-mode protein chromatography: Principles, characteristics and design. , 2009, Journal of biotechnology.

[156]  B. P. Tripathi,et al.  Membrane-based techniques for the separation and purification of proteins: an overview. , 2009, Advances in colloid and interface science.

[157]  Yun Kang,et al.  Chromatographic removal of endotoxin from hemoglobin preparations. Effects of solution conditions on endotoxin removal efficiency and protein recovery. , 1998, Journal of chromatography. A.

[158]  T. Gunter,et al.  High-yield expression and purification of p18 form of Bax as an MBP-fusion protein. , 2004, Protein expression and purification.

[159]  Juan A. Asenjo,et al.  Optimization and design considerations of two‐phase continuous protein separation , 1999 .

[160]  Joe D. Cohen,et al.  Phase I safety and immunogenicity trial of FMP1/AS02A, a Plasmodium falciparum MSP-1 asexual blood stage vaccine. , 2006, Vaccine.

[161]  Etsuo Shinohara,et al.  Free-flow electrophoresis in a microfabricated chamber with a micromodule fraction separator. Continuous separation of proteins. , 2003, Journal of chromatography. A.

[162]  J. Asenjo,et al.  Methodology for predicting the separation of proteins by hydrophobic interaction chromatography and its application to a cell extract. , 2003, Journal of chromatography. A.

[163]  S. Welling-Wester,et al.  Combined size-exclusion and reversed-phase high-performance liquid chromatography of a detergent extract of Sendai virus. , 1985, Journal of chromatography.

[164]  Joseph J. Falke,et al.  [16] Purification of proteins using polyhistidine affinity tags , 2000 .

[165]  R. Stewart,et al.  Purification of human immunoglobulin G: a new approach to plasma fractionation , 2002, Vox sanguinis.

[166]  Daniel I. C. Wang,et al.  Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 2. Effect of entrained micelle-poor domains. , 2002, Biotechnology and bioengineering.

[167]  Arne Skerra,et al.  The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins , 2007, Nature Protocols.

[168]  C. Lowe,et al.  Combinatorial approaches to affinity chromatography. , 2001, Current opinion in chemical biology.

[169]  A. Seidel-Morgenstern,et al.  Discontinuous and continuous purification of single-chain antibody fragments using immobilized metal ion affinity chromatography. , 2013, Journal of biotechnology.

[170]  M. Morbidelli,et al.  Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP) , 2010, Biotechnology and bioengineering.

[171]  G. Jackowski,et al.  Removal of endotoxin from recombinant protein preparations. , 1997, Clinical Biochemistry.