Multimodal or Mixed-Mode Chromatography

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en-us MWHB6KKSY Multimodal or Mixed-Mode Chromatography Multimodal or Mixed-Mode Chromatography /webroot/web/html/lsr/solutions/technologies/chromatography <p>Multimodal or mixed-mode protein chromatography is based on media supports that have been functionalized with ligands capable of multiple modes of interaction: ion exchange, hydroxyapatite, affinity, size exclusion, and hydrophobic interactions. The ability to combine these separation methods can enhance selectivity in a protein purification process. This section provides an overview of multimodal chromatography, discusses some general considerations for mixed-mode chromatography, and describes hydrophobic ion-exchange and mixed-mode media and ligands.</p> <p>To find out whether multimodal chromatography is the most suitable approach for your application and to learn about other chromatographic methods that may be applicable for your purification needs, visit our <a href="/evportal/destination/solutions?catID=LUSMIS7OP">Chromatography</a> page.</p> <p><strong> Related Topics:</strong> <a href="/en-us/applications-technologies/liquid-chromatography/size-exclusion-chromatography">Size Exclusion Chromatography</a>, <a href="/en-us/applications-technologies/liquid-chromatography-principles/ion-exchange-chromatography">Ion Exchange Chromatography</a>, <a href="/evportal/destination/solutions?catID=MWHB53MNI">Hydrophobic Interaction Chromatography</a>, <a href="http://qaevn.bio-rad.com/en-us/applications-technologies/liquid-chromatography/affinity-chromatography">Affinity Chromatography</a>, <a href="/evportal/destination/solutions?catID=MWHBAYLPT">Low Pressure Chromatography Systems</a> and <a href="/evportal/destination/solutions?catID=MWHBD3HYP">Medium Pressure Chromatography Systems</a>.</p> General Considerations <p>Unlike affinity chromatography where a specific site on the protein is targeted, with mixed-mode ligands there is no known specificity. Accordingly, screening mixed-mode media becomes a search for sites on the target protein that will provide useful affinity and selectivity.</p> <p>Mixed-mode chromatography interactions are not independent of one another. For example, when using a mixed-mode ligand containing both hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds, however, increasing salt concentration will promote hydrophobic interactions.</p> <p>Mixed-mode media effectively combine complementary chromatography methods within a single media and can reduce the total number of column steps needed in a purification process. Because these mixed-mode elements are present in a single ligand, it contributes affinity-like binding and selectivity.</p> <p>Binding and elution are controlled and optimized by the parameters relevant to each mode &mdash; salt for hydrophobic interactions and ionic strength for ionic interactions.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Mixed-Mode Media <p>There are a number of commercially available mixed-mode media combining different chromatographic elements:</p> <ul> <li>Hydroxyapatite (CHT/CFT/Bio-Gel HT/HTP) &mdash; electrostatic and calcium coordination complexes</li> <li>Hydrophobic ion exchange ligands</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Hydroxyapatite Chromatography <p>Hydroxyapatite, Ca<sub>10</sub>(PO<sub>4</sub>)<sub>6</sub>(OH)<sub>2</sub>, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of &ndash;OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques.</p> <p>Applications of hydroxyapatite chromatography include the purification of:</p> <ul> <li>Different subclasses of monoclonal and polyclonal antibodies</li> <li>Antibodies that differ in light chain composition</li> <li>Antibody fragments</li> <li>Recombinant proteins </li> <li>Viral particles</li> <li>Vaccines</li> <li>Isozymes</li> <li>Supercoiled DNA from linear duplexes</li> <li>Single-stranded from double-stranded DNA</li> </ul> <p>CHT&trade; ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a nanocrystalline to a ceramic form. The ceramic material retains the unique separation properties of crystalline hydroxyapatite, and lot-to-lot control assures reproducibility in large-scale production columns. Unlike most other chromatography adsorbents, CHT is both the ligand and the support matrix. Separation protocols originally developed on crystalline hydroxyapatite can often be transferred directly to the ceramic material with only minor modifications.</p> <p>Two types of CHT ceramic hydroxyapatite, <a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=34cbb522-888f-43c7-aaf5-a61cfada935e">Type I</a> and <a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=a642d16e-5ca8-4981-acf2-a57271984632">Type II</a>, are available in three particle sizes, 20, 40, and 80 &mu;m. Although both types have elution characteristics similar to crystalline hydroxyapatite, there are also some important differences. CHT Type I has a higher protein binding capacity and better capacity for acidic proteins. CHT Type II has a lower protein binding capacity but has better resolution of nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/lab_scale_chromatography/chromatography/technology_detail/chromt15_img1.jpg" alt="" width="352" height="205" /></p> <p class="caption" style="width: 352px;"><strong>Protein binding to hydroxyapatite.</strong> <strong>A</strong> is a basic protein. <strong>B</strong> is an acidic protein. Double parentheses indicate repulsion. Dotted lines indicate ionic bonds. Triangular linkages indicate coordination bonds.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Hydrophobic Ion-Exchange Ligands <p>These ligands incorporate hydrophobic and ionic elements. For ion exchange binding and elution, parameters such as salt, pH, ionic strength, and buffer apply. With hydrophobic interactions, salt type, concentration, and additives apply. As mentioned above, it is important to remember that binding and elution factors are not independent and can work counter to one another. For example, increasing the ionic strength for ion-exchange elution will drive hydrophobic binding. Due to the dependency of these interactions, buffer conditions must be optimized for binding, washing, and elution to determine the optimal balance for a highly selective purification scheme.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Mixed-Mode Ligands <ul> <li><strong>Hydrophobic, Anionic Ligand with Hydrogen Bonding</strong> &mdash; this ligand features hydrogen bonding, a quaternary amine, and a phenyl group</li> <li><strong>Mixed-Mode Cationic Ligand with Hydrophobic Binding</strong> &mdash; this ligand contains a secondary amine and is cationic over a wide pH range, therefore, it behaves as both a hydrophobic interaction resin and an anionic exchange media. At low ionic strength, it can bind acidic proteins as well as proteins with moderately high isoelectric points. However, hydrophobic interactions predominate, as binding capacity increases with temperature and salt</li> <li><strong>Mixed-Mode pH-Controllable Sorbents</strong> &mdash; this ligand contains a 4-mercaptoethylpyridine (MEP) ligand. The pyridine ring is uncharged at neutral and basic pH. As the pH decreases, the pyridine nitrogen becomes positively charged, turning the resin into a mixed-mode media. MEP becomes a pH-controlled mixed-mode ligand, a property that has been termed "hydrophobic charge induction chromatography" (Burton and Harding, 1998)</li> </ul> <p><img style="float: left; margin-right:10px;" src="/webroot/web/images/lsr/solutions/technologies/lab_scale_chromatography/chromatography/technology_detail/chromt15_img2.jpg" alt="" width="263" height="229" /></p> <p class="caption" style="margin-left:20px; margin-top:85px"><strong>Example of mixed-mode ligands. A</strong> is a hydrophobic, anionic ligand with hydrogen bonding. <strong>B</strong> is a mixed-mode pH-controllable sorbent. <strong>C</strong> is a mixed-mode cationic ligand with hydrophobic binding.</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <div class="top"><a href="#helptop">Back to Top</a></div> Mixed Mode Chromatography Media Selection Guide <table class="pd_table" border="0"> <tbody> <tr> <td class="bordertop" colspan="2" valign="top">&nbsp;</td> <td class="bordertop borderbttm" colspan="3" align="center" valign="top"><strong>Suitability**</strong></td> <td class="bordertop" valign="top">&nbsp;</td> </tr> <tr> <td valign="bottom"><strong>Media Type</strong></td> <td valign="bottom"><strong>Packaging<br /> Format*</strong></td> <td valign="bottom"><strong>Analytical<br /> Scale</strong></td> <td valign="bottom"><strong>Pilot/<br />Preparative<br />Scale</strong></td> <td valign="bottom"><strong>Process<br /> Scale</strong></td> <td valign="bottom"><strong>Application</strong></td> </tr> <tr> <td class="bordertop" colspan="6" valign="top"><strong>Hydroxyapatite and Fluoroapatite</strong></td> </tr> <tr class="pd_colorbackground"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=7b9e4566-4a07-482a-8082-62dfcb1dd0b1">CHT&trade;<br />Type I</a></td> <td valign="top">B, C, MPC</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">Antibody purification (higher capacity than Type II); virus purification/removal; DNA purification/removal; aggregate and endotoxin removal; Fab purification</td> </tr> <tr class="pd_table pd_gridlines"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=5ded4ec5-ff5a-47b7-bf10-07253005c83f">CHT<br />Type II</a></td> <td valign="top">B, C</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">Antibody purification; removal of albumin from feedstream; Fab purification</td> </tr> <tr class="pd_colorbackground"> <td valign="top">CFT&trade;<br />Types I and II</td> <td valign="top">B, C</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">Similar properties to CHT but exhibits greater stability in the lower pH range (5.5); suitable for Fab purification</td> </tr> <tr class="pd_table pd_gridlines"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=ea60717e-e888-4b44-ac03-61600278c77c">Bio-Gel<sup>&reg;</sup> HT</a></td> <td valign="top">B</td> <td valign="top">++++</td> <td valign="top">+++</td> <td valign="top">&nbsp;</td> <td valign="top">Purification of proteins, nucleic acids and other biomolecules; crystalline hydroxyapatite not as mechanically stable as CHT (ceramic hydroxyapatite)</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=57f66f33-143c-402c-bde8-8cbd9ceebdb4">Bio-Gel<sup>&reg;</sup> HTP</a></td> <td valign="top">B</td> <td valign="top">++++</td> <td valign="top">+++</td> <td valign="top">&nbsp;</td> <td valign="top">Similar to Bio-Gel HT but in powder form</td> </tr> <tr class="pd_table pd_gridlines"> <td valign="top">DNA grade Bio-Gel<sup>&reg;</sup> HTP</td> <td valign="top">B</td> <td valign="top">++++</td> <td valign="top">+++</td> <td valign="top">&nbsp;</td> <td valign="top">Similar to Bio-Gel HTP with smaller particle size; selectivity for dsDNA; separation of ss- and dsDNA</td> </tr> </tbody> </table> <p class="caption">* B, bottle; C, cartridge (1 ml or 5 ml); GC, gravity column; SC, spin column; HPLC, high-pressure column; MPC, medium-pressure column.</p> <p class="caption">** +, low suitability; ++, moderate suitability; +++, suitable; ++++, high suitability.</p> <div class="top"><a href="#helptop">Back to Top</a></div> References <p>Burton SC and Harding DR (1998). Hydrophobic charge induction chromatography: Salt independent protein adsorption and facile elution with aqueous buffers. J Chromatogr A 814, 71&ndash;81.</p> <div class="top"><a href="#helptop">Back to Top</a></div> 5709 /templatedata/internet/documentation/data/LSR/Literature/5709_0910141009835.xml 5709 How CHT Ceramic Hydroxyapatite Works, Rev A H /webroot/web/pdf/psd/literature/BULLETIN_5709A.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No How CHT Ceramic Hydroxyapatite Works, Rev A Life Science 5709 1582000, 1588000, LIT5709, 158-2000, 1584200, protein separation, 158-4200, 158-8000, Bio-Scale Mini CHT-I cartridge, bioscale CHT-II cartridges, ion exchange, Bulletin 5709, 158-2200, metal affinity chromatography, 1582200, 1588200, 158-4000, 158-8200, 158400 6086 /templatedata/internet/documentation/data/LSR/Literature/6086.xml Ceramic Hydroxyapatite Application Guide for Process Development and Scale-Up 6086 H /webroot/web/pdf/lsr/literature/Bulletin_6086.pdf Literature PDF Manuals_and_Quick_Guides /webroot/web/images/general/icons/icon_pdf.gif No Ceramic Hydroxyapatite Application Guide for Process Development and Scale-Up 6086 Bulletin 6086, CHT, Ceramic hydroxyapatite, Type I and type II media, support, CHT-packed bed, protein and monoclonal antibody capture and purification protocols, mAb, immunoglobulin, IgG, sintered calcium phosphate, high or low sample buffering capacity, contaminant removal, column packing and unpacking techniques; open column; closed column; chromatography, binding capacity, column volumes, CV; regeneration, sanitation and storage; slurry mixing, slurry transfer, 158-2200, 1582200, 157-2000, 1572000, 157-2100, 1572100, 157-2500, 1572500, 158-4200, 1584200, 158-4000, 1584000, 157-4100, 1574100, 157-4500, 1574500, 158-8200, 1588200, 157-8000, 1578000, 157-8100, 1578100, 157-8500, 1578500, 158-2000, 1582000, 157-0020, 1570020, 157-0021, 1570021, 157-0025, 1570025, 157-0040, 1570040, 157-0041, 1570041, 157-0045, 1570045, 158-8000, 1588000, 157-0080, 1570080, 157-0081, 1570081, 157-0085, 1570085, CHT instruction manual, CHT application guide, Lit611, Lit 611, Lit-611, cation exchange, metal affinity, mixed-mode 5667 /templatedata/internet/documentation/data/LSR/Literature/5667_0910141009863.xml CHT Ceramic Hydroxyapatite Product Information Sheet, Rev B 5667 H /webroot/web/pdf/psd/literature/Bulletin_5667.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif CHT Ceramic Hydroxyapatite Product Information Sheet, Rev B No Ceramic Hydroxyapatite Product Information Sheet, Rev B 5667 5667, chromatography, column, columns, media, resin, mpc, cht, cft, hydroxyapatite, hydroxyfluoroapatite, 157-0020, 157-0021, 157-0025, 157-0040, 157-0041, 157-0045, 157-0080, 157-0081, 157-0085, 157-2000, 157-2100, 157-2500, 157-4000, 157-4100, 157-4500, 157-8000, 157-8100, 157-8500, 158-2000, 158-2000EDU, 158-2200, 158-4000, 158-4200, 158-8000, 158-8200, 1570020, 1570021, 1570025, 1570040, 1570041, 1570045, 1570080, 1570081, 1570085, 1572000, 1572100, 1572500, 1574000, 1574100, 1574500, 1578000, 1578100, 1578500, 1582000, 1582000EDU, 1582200, 1584000, 1584200, 1588000, 1588200 2156 /templatedata/internet/documentation/data/LSR/Literature/2156_0910140920541.xml CHT Ceramic Hydroxyapatite &#151; A New Dimension in Chromatography of Biological Molecules, Rev C 2156 /webroot/web/pdf/psd/literature/Bulletin_2156.pdf Literature PDF Product_Information_Sheets /webroot/web/images/general/icons/icon_pdf.gif No CHT Ceramic Hydroxyapatite &#151; A New Dimension in Chromatography of Biological Molecules, Rev C 2156 bulletin 2156, ht, process separation, separation, macroprep, fractionation, lit2156, macro-prep, proteomeworks, htp, protein purification, sample preparation, mixed mode, mixed-mode, dual, multimode, multimodal, cht, cft, ceramic, hydroxyapatite, fluoroapatite, chromatography, column, columns, medium, media, resin, resins, protein, purification, metal affiinity, ion exchange, 158-2000, 158-4000, 157-4000, 157-0040, 158-4200, 157-0020, 158-8000, 157-8000, 158-8200, 157-0080, 157-2000, 158-2200, 157-0045, 157-0041, 157-0021, 157-2100, 157-8500, 157-4100, 157-4500, 157-0081, 157-8100, 157-0025, 157-0085, 157-2500, 1582000EDU, 1582000, 1584000, 1574000, 1570040, 1584200, 1570020, 1588000, 1578000, 1588200, 1570080, 1572000, 1582200, 1570045, 1570041, 1570021, 1572100, 1578500, 1574100, 1574500, 1570081, 1578100, 1570025, 1570085, 1572500, 1582000EDU 6601 /templatedata/internet/documentation/data/LSR/Literature/6601_1411507329.xml Enhancing the Flexibility of the NGC&#153; Chromatography System: Addition of a Refractive Index Detector for Wine Sample Analysis, Rev A 6601 H /webroot/web/pdf/lsr/literature/Bulletin_6601.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif Enhancing the Flexibility of the NGC Chromatography System: Addition of a Refractive Index Detector for Wine Sample Analysis No Enhancing the Flexibility of the NGC Chromatography System: Addition of a Refractive Index Detector for Wine Sample Analysis 6601 6601, Aminex HPX-87H column, HPLC, mixed mode, ion exclusion, multi-wavelength detector, Quest Plus, F10 gradient pump, ChromLab software, separation, complex sample, 788-0003, 7880003, 125-0140, 1250140 1801 /templatedata/internet/documentation/data/LSR/Literature/1801_0910140920099.xml Protein and Peptide Purification Applications, Rev B 1801 /webroot/web/pdf/lsr/literature/Bulletin_1801.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Protein and Peptide Purification Applications, Rev B 1801 lit1801, bulletin 1801, chromatography, column, columns, medium, media, resin, resins, protein, purification, affinity, profinity, profinia, ion, exchange, iex, anion, cation, anex, catex, cex, mixed mode, mixed-mode, dual, multimode, multimodal, cht, cft, hydroxyapatite, fluoroapatite, hic, hydrophobic, size exclusion, sec, gpc, gel permeation, desalting, applications 6067 /templatedata/internet/documentation/data/LSR/Literature/6067.xml EDTA Complexometric Titration of Hydroxyapatite Column Effluent, Rev B 6067 /webroot/web/pdf/lsr/literature/Bulletin_6067.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif EDTA Complexometric Titration of Hydroxyapatite Column Effluent, Rev B No EDTA Complexometric Titration of Hydroxyapatite Column Effluent, Rev B 6067 ceramic hydroxyapatite, cht, packed bed, protein and monoclonal antibody purification protocols, sintered calcium phosphate, high or low sample buffering capacity, calcium, hydroxyapatite, edta, titration, 158-2200, 1582200, 157-2000, 1572000, 157-2100, 1572100, 157-2500, 1572500, 158-4200, 1584200, 158-4000, 1584000, 157-4100, 1574100, 157-4500, 1574500, 158-8200, 1588200, 157-8000, 1578000, 157-8100, 1578100, 157-8500, 1578500, 158-2000, 1582000, 157-00, 161-0729, 1610729 RP0033 /templatedata/internet/documentation/data/PSD/Literature/RP0033.xml A Ceramic Hydroxyapatite–Based Purification Platform &mdash; Simultaneous Removal of Leached Protein A, Aggregates, DNA, and Endotoxins from MAbs RP0033 H /webroot/web/pdf/ps/literature/Bulletin_RP0033.pdf Literature PDF Articles_and_Whitepapers /webroot/web/images/general/icons/icon_pdf.gif No A Ceramic Hydroxyapatite–Based Purification Platform &mdash; Simultaneous Removal of Leached Protein A, Aggregates, DNA, and Endotoxins from MAbs RP0033 2524 /templatedata/internet/documentation/data/LSR/Literature/2524_0910140920029.xml 2524 Purification of Horse IgG<sub>T</sub> Using Macro-Prep DEAE and CHT Ceramic Hydroxyapatite Type I Supports, Rev C /webroot/web/pdf/lsr/literature/Bulletin_2524C.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No Purification of Horse IgG<sub>T</sub> Using Macro-Prep DEAE and CHT Ceramic Hydroxyapatite Type I Supports, Rev C Life Science 2524 immunoglobulins, Bio-Scale, chromatographic, columns, column, LIT2524, bulletin 2524, Igg, immunoglobulin 2774 /templatedata/internet/documentation/data/LSR/Literature/2774_0910140920158.xml 2774 Purification of Transgenic Antibody From Corn Seed Using UNOsphere S and CHT Ceramic Hydroxyapatite Supports, Rev A /webroot/web/pdf/lsr/literature/Bulletin_2774.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No Purification of Transgenic Antibody From Corn Seed Using UNOsphere S and CHT Ceramic Hydroxyapatite Supports, Rev A Life Science 2774 unosphere, ceramic hydroxyapapatite, bulletin 2774, cht, LIT2774 5853 /templatedata/internet/documentation/data/LSR/Literature/5853_0910141008991.xml Chimeric IgG Monoclonal Antibody Purification: A Comparative Study Using CHT Ceramic Hydroxyapatite and CFT Ceramic Fluoroapatite Chromatographic Media, Rev A 5853 /webroot/web/pdf/lsr/literature/Bulletin_5853.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Chimeric IgG Monoclonal Antibody Purification: A Comparative Study Using CHT Ceramic Hydroxyapatite and CFT Ceramic Fluoroapatite Chromatographic Media, Rev A 5853 Mab, biopharmaceutical manufacturing, protein A, multimodal chromatography support, UNOsphere SUPrA medium, affinity, LIT5853, mixed mode, tech notes, bulletin 5853 6549 /templatedata/internet/documentation/data/LSR/Literature/6549_1411507790.xml Mammalian Virus Purification Using Ceramic Hydroxyapatite, Rev A 6549 H /webroot/web/pdf/lsr/literature/Bulletin_6549.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif Mammalian Virus Purification Using Ceramic Hydroxyapatite No Mammalian Virus Purification Using Ceramic Hydroxyapatite 6549 6549, CHT resin, media, CFT, MPC, chromatography, BioLogic DuoFlow System, separation, process intermediates, vaccine production, flow rate, gradient slope 6278 /templatedata/internet/documentation/data/PSD/Literature/6278.xml High-Throughput Calcium Analysis of Hydroxyapatite Column Effluent, Rev A 6278 H /webroot/web/pdf/psd/literature/Bulletin_6278.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif High-Throughput Calcium Analysis of Hydroxyapatite Column Effluent, Rev A No High-Throughput Calcium Analysis of Hydroxyapatite Column Effluent, Rev A 6278 6278, ceramic hydroxyapatite, CHT, mAb, mixed-mode media, monoclonal antibody purification, multimodal media, process chromatography, protein purification, purification protocol 2731 /templatedata/internet/documentation/data/LSR/Literature/2731_0910140920376.xml Plasmid Purification Using CHT Ceramic Hydroxyapatite Support, Rev A 2731 /webroot/web/pdf/psd/literature/BULLETIN_2731.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Plasmid Purification Using CHT Ceramic Hydroxyapatite Support, Rev A 2731 lysates, macroprep, media, contaminant removal, LIT2731, medium, sample preparation, transfer, bulletin 2731, plasmids, macro-prep, dna purification, gene delivery, lysate, prep, chromatography support, isolation, scalable method 2780 /templatedata/internet/documentation/data/LSR/Literature/2780_0910140919395.xml Purification of Murine IgG<sub>1</sub> Using UNOsphere S and CHT Ceramic Hydroxyapatite Chromatography, Rev A 2780 /webroot/web/pdf/lsr/literature/Bulletin_2780.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Purification of Murine IgG<sub>1</sub> Using UNOsphere S and CHT Ceramic Hydroxyapatite Chromatography, Rev A 2780 Uno Sphere, LIT2780, bulletin 2780 1986 /templatedata/internet/documentation/data/LSR/Literature/1986_0910140920404.xml Effect of pH on Gradient Elution of Proteins on Two Types of CHT Ceramic Hydroxyapatite, Rev B 1986 H /webroot/web/pdf/lsr/literature/Bulletin_1986B.pdf Literature PDF Scientific_Posters /webroot/web/images/general/icons/icon_pdf.gif No Effect of pH on Gradient Elution of Proteins on Two Types of CHT Ceramic Hydroxyapatite, Rev B 1986 chromatography, column, columns, medium, media, resin, resins, protein, purification, affinity, metal, ion, exchange, iex, anion, cation, anex, catex, cex, mixed mode, mixed-mode, dual, multimode, multimodal, cht, cft, ceramic, hydroxyapatite, fluoroapatite, gradient, ph 2940 /templatedata/internet/documentation/data/LSR/Literature/2940_0910140920460.xml Removal of Aggregate from an IgG<sub>4</sub> Product Using CHT&trade; Ceramic Hydroxyapatite Resin, Rev B 2940 /webroot/web/pdf/psd/literature/Bulletin_2940.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif Removal of Aggregate from an IgG<sub>4</sub> Product Using CHT&trade; Ceramic Hydroxyapatite Resin No Removal of Aggregate from an IgG<sub>4</sub> Product Using CHT&trade; Ceramic Hydroxyapatite Resin, Rev B 2940 bulletin 2940, lit2940, antibody, purification, 2940, cht, ceramic hydroxyapatite, bio-sil, biosil, biologic duoflow, bio-scale mt, bioscale mt, aggregate, aggregation, impurities, immunoglobulin, monomer, size exclusion chromatography, sec, igg, igg4, 158-2000, 125-0062, 760-0037, 751-0081, 1582000, 1250062, 7600037, 7510081 2849 /templatedata/internet/documentation/data/LSR/Literature/2849_0910140919369.xml 2849 Protein A Removal From IgG on CHT Ceramic Hydroxyapatite Support, Rev A /webroot/web/pdf/lsr/literature/Bulletin_2849.pdf Literature PDF Application Notes /webroot/web/images/general/icons/icon_pdf.gif No Protein A Removal From IgG on CHT Ceramic Hydroxyapatite Support, Rev A Life Science 2849 LIT2849, bulletin 2849, protein purification 2535 /templatedata/internet/documentation/data/LSR/Literature/2535_0910140920431.xml Purification of Recombinant Human alpha 1-Antitrypsin From Transgenic Milk Using CHT Ceramic Hydroxyapatite, Rev B 2535 /webroot/web/pdf/psd/literature/Bulletin_2535.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Purification of Recombinant Human alpha 1-Antitrypsin From Transgenic Milk Using CHT Ceramic Hydroxyapatite, Rev B 2535 bulletin 2535, LIT2535, protein 2575 /templatedata/internet/documentation/data/LSR/Literature/2575_0910140920488.xml Purification of a Calcium-Binding Protein Using CHT Ceramic Hydroxyapatite 2575 /webroot/web/pdf/psd/literature/Bulletin_2575.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Purification of a Calcium-Binding Protein Using CHT Ceramic Hydroxyapatite 2575 bulletin 2575, LIT2575 2946 /templatedata/internet/documentation/data/LSR/Literature/2946_0910140920515.xml Plasmid DNA Adsorption Behavior on CHT Ceramic Hydroxyapatite Type II Support, Rev A 2946 /webroot/web/pdf/psd/literature/Bulletin_2946.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Plasmid DNA Adsorption Behavior on CHT Ceramic Hydroxyapatite Type II Support, Rev A 2946 sample preparation, bulletin 2946, LIT2946 5831 /templatedata/internet/documentation/data/LSR/Literature/5831_0910141009806.xml Process-scale packing procedures for CHT ceramic hydroxyapatite in open column systems: Best practices, Rev A 5831 /webroot/web/pdf/psd/literature/Bulletin_5831.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Process-scale packing procedures for CHT ceramic hydroxyapatite in open column systems: Best practices, Rev A 5831 Process scale chromatography columns, easy pack, 157-4200, 1588000, biopharmaceutical manufacturing, slurry, 157-8200, 1570081, 1578200, 1588200, bulletin 5831, pilot-scale, 157-0040, 157-0081, 157-8100, 1574100, asymmetry test, easypack, pilot, 157-0041, 157-4100, 1570041, 1584000, LIT5831, 157-0045, 157-0080, 157-0085, 157-8000, 1570080, 1574000, 1574200, 1578000, 158-4200, 158-8000, packed bed, tech note, 1570085, 1578100, 1584200, HETP testing, slurrying, 157-4000, 1570040, 1570045, 158-4000, 158-8200 5825 /templatedata/internet/documentation/data/LSR/Literature/5825_0910141009892.xml 5913 /templatedata/internet/documentation/data/PSD/Literature/5913_1256598170.xml Separation of Fab and Fc Fragments from Monoclonal Antibody Papain Digest on Ceramic Hydroxyapatite and Ceramic Fluoroapatite, Rev A 5913 /webroot/web/pdf/ps/literature/Bulletin_5913.pdf Literature PDF Scientific_Posters /webroot/web/images/general/icons/icon_pdf.gif No 5913 Mab fragment, therapeutic agent, biotherapeutic agents, biopharmaceutical, biopharmaceuticals, protein A affinity, protein G, ion exchange, gel filtration chromatography, CHT, CFT, separations, bulletin 5913, size exclusion, 158-2000, 158-2200, 158-4000, 158-4200,157-2000, 157-0020, 157-0021, 157-2100, 157-2500, 157-0025, 157-4000, 157-0040, 157-4100, 157-0041, 157-0045, 157-4500, 1582000, 1582200, 1584000, 1584200,1572000, 1570020, 1570021, 1572100, 1572500, 1570025, 1574000, 1570040, 1574100, 1570041, 1570045, 1574500,158-5200, 157-5000, 157-5100,157-5500, 1585200, 1575000, 1575100,1575500 6749 /templatedata/internet/documentation/data/LSR/Literature/6749_1450213106.xml Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes, Ver A 6749 H /webroot/web/pdf/lsr/literature/Bulletin_6749.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes No Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes 6749 6749, process separations, protein purification, chromatography, cht, ceramic hydroxyapatite, monoclonal antibodies, mab s, monomer, aggregate removal, media, resin, mixed-mode, mixed mode, protein a affinity, size exclusion, bio-scale mini cartridge, bio scale, bioscale, enrich sec 650, ngc, ngc 10, 1584000, 1570040, 1570041, 1570045, 157-0041, 157-0045, 7801650 Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/Deionizing Analytical Grade Resins ->MT::803a59da-012b-4afb-b0dc-e64e1d5e7d66##Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/CHT Ceramic Hydroxyapatite Type I Resin ->MT::34cbb522-888f-43c7-aaf5-a61cfada935e##Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/CHT Ceramic Hydroxyapatite Type II Resin ->MT::a642d16e-5ca8-4981-acf2-a57271984632## Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/Bio-Gel Hydroxyapatite HT and HTP Media ->MT::1e754042-4d9d-4487-ba7b-dc67dec9f8fc##Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/Fluoroapatite Media ->MT::f6456568-f543-48d2-a210-b727ab3137e8## Life Science Research/Solutions/Technologies/Protein Electrophoresis/Protein Sample Preparation ->MTS::LUSP108WI##Life Science Research/Solutions/Applications/Automated Electrophoresis and Analysis ->MTS::LUSQXB15##Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Applications/Protein Interaction Analysis ->MTS::LUSLTJE8Z##Life Science Research/Solutions/Technologies/Surface Plasmon Resonance ->MTS::LUSM664EH## Karen Moss Multimodal or Mixed-Mode Chromatography 11/20/13 01:26 PM 11/20/23 01:40 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/Chromatography N 0 Liquid Chromatography Principles /en-us/applications-technologies/applications-technologies/multimodal-or-mixed-mode-chromatography?ID=MWHAS7E8Z

Multimodal or mixed-mode protein chromatography is based on media supports that have been functionalized with ligands capable of multiple modes of interaction: ion exchange, hydroxyapatite, affinity, size exclusion, and hydrophobic interactions. The ability to combine these separation methods can enhance selectivity in a protein purification process. This section provides an overview of multimodal chromatography, discusses some general considerations for mixed-mode chromatography, and describes hydrophobic ion-exchange and mixed-mode media and ligands.

To find out whether multimodal chromatography is the most suitable approach for your application and to learn about other chromatographic methods that may be applicable for your purification needs, visit our Chromatography page.

Related Topics: Size Exclusion Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction Chromatography, Affinity Chromatography, Low Pressure Chromatography Systems and Medium Pressure Chromatography Systems.

 

General Considerations

Unlike affinity chromatography where a specific site on the protein is targeted, with mixed-mode ligands there is no known specificity. Accordingly, screening mixed-mode media becomes a search for sites on the target protein that will provide useful affinity and selectivity.

Mixed-mode chromatography interactions are not independent of one another. For example, when using a mixed-mode ligand containing both hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds, however, increasing salt concentration will promote hydrophobic interactions.

Mixed-mode media effectively combine complementary chromatography methods within a single media and can reduce the total number of column steps needed in a purification process. Because these mixed-mode elements are present in a single ligand, it contributes affinity-like binding and selectivity.

Binding and elution are controlled and optimized by the parameters relevant to each mode — salt for hydrophobic interactions and ionic strength for ionic interactions.

 

Mixed-Mode Media

There are a number of commercially available mixed-mode media combining different chromatographic elements:

  • Hydroxyapatite (CHT/CFT/Bio-Gel HT/HTP) — electrostatic and calcium coordination complexes
  • Hydrophobic ion exchange ligands
 

Hydroxyapatite Chromatography

Hydroxyapatite, Ca10(PO4)6(OH)2, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of –OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques.

Applications of hydroxyapatite chromatography include the purification of:

  • Different subclasses of monoclonal and polyclonal antibodies
  • Antibodies that differ in light chain composition
  • Antibody fragments
  • Recombinant proteins
  • Viral particles
  • Vaccines
  • Isozymes
  • Supercoiled DNA from linear duplexes
  • Single-stranded from double-stranded DNA

CHT™ ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a nanocrystalline to a ceramic form. The ceramic material retains the unique separation properties of crystalline hydroxyapatite, and lot-to-lot control assures reproducibility in large-scale production columns. Unlike most other chromatography adsorbents, CHT is both the ligand and the support matrix. Separation protocols originally developed on crystalline hydroxyapatite can often be transferred directly to the ceramic material with only minor modifications.

Two types of CHT ceramic hydroxyapatite, Type I and Type II, are available in three particle sizes, 20, 40, and 80 μm. Although both types have elution characteristics similar to crystalline hydroxyapatite, there are also some important differences. CHT Type I has a higher protein binding capacity and better capacity for acidic proteins. CHT Type II has a lower protein binding capacity but has better resolution of nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.

Protein binding to hydroxyapatite. A is a basic protein. B is an acidic protein. Double parentheses indicate repulsion. Dotted lines indicate ionic bonds. Triangular linkages indicate coordination bonds.

 

Hydrophobic Ion-Exchange Ligands

These ligands incorporate hydrophobic and ionic elements. For ion exchange binding and elution, parameters such as salt, pH, ionic strength, and buffer apply. With hydrophobic interactions, salt type, concentration, and additives apply. As mentioned above, it is important to remember that binding and elution factors are not independent and can work counter to one another. For example, increasing the ionic strength for ion-exchange elution will drive hydrophobic binding. Due to the dependency of these interactions, buffer conditions must be optimized for binding, washing, and elution to determine the optimal balance for a highly selective purification scheme.

 

Mixed-Mode Ligands

  • Hydrophobic, Anionic Ligand with Hydrogen Bonding — this ligand features hydrogen bonding, a quaternary amine, and a phenyl group
  • Mixed-Mode Cationic Ligand with Hydrophobic Binding — this ligand contains a secondary amine and is cationic over a wide pH range, therefore, it behaves as both a hydrophobic interaction resin and an anionic exchange media. At low ionic strength, it can bind acidic proteins as well as proteins with moderately high isoelectric points. However, hydrophobic interactions predominate, as binding capacity increases with temperature and salt
  • Mixed-Mode pH-Controllable Sorbents — this ligand contains a 4-mercaptoethylpyridine (MEP) ligand. The pyridine ring is uncharged at neutral and basic pH. As the pH decreases, the pyridine nitrogen becomes positively charged, turning the resin into a mixed-mode media. MEP becomes a pH-controlled mixed-mode ligand, a property that has been termed "hydrophobic charge induction chromatography" (Burton and Harding, 1998)

Example of mixed-mode ligands. A is a hydrophobic, anionic ligand with hydrogen bonding. B is a mixed-mode pH-controllable sorbent. C is a mixed-mode cationic ligand with hydrophobic binding.

 

 

 

 

Mixed Mode Chromatography Media Selection Guide

  Suitability**  
Media Type Packaging
Format*
Analytical
Scale
Pilot/
Preparative
Scale
Process
Scale
Application
Hydroxyapatite and Fluoroapatite
CHT™
Type I
B, C, MPC ++++ ++++ ++++ Antibody purification (higher capacity than Type II); virus purification/removal; DNA purification/removal; aggregate and endotoxin removal; Fab purification
CHT
Type II
B, C ++++ ++++ ++++ Antibody purification; removal of albumin from feedstream; Fab purification
CFT™
Types I and II
B, C ++++ ++++ ++++ Similar properties to CHT but exhibits greater stability in the lower pH range (5.5); suitable for Fab purification
Bio-Gel® HT B ++++ +++   Purification of proteins, nucleic acids and other biomolecules; crystalline hydroxyapatite not as mechanically stable as CHT (ceramic hydroxyapatite)
Bio-Gel® HTP B ++++ +++   Similar to Bio-Gel HT but in powder form
DNA grade Bio-Gel® HTP B ++++ +++   Similar to Bio-Gel HTP with smaller particle size; selectivity for dsDNA; separation of ss- and dsDNA

* B, bottle; C, cartridge (1 ml or 5 ml); GC, gravity column; SC, spin column; HPLC, high-pressure column; MPC, medium-pressure column.

** +, low suitability; ++, moderate suitability; +++, suitable; ++++, high suitability.

 

References

Burton SC and Harding DR (1998). Hydrophobic charge induction chromatography: Salt independent protein adsorption and facile elution with aqueous buffers. J Chromatogr A 814, 71–81.

 

Related Content

 
Literature
Number Description Download
5709 How CHT Ceramic Hydroxyapatite Works, Rev A Click to download
6086 Ceramic Hydroxyapatite Application Guide for Process Development and Scale-Up Click to download
5667 CHT Ceramic Hydroxyapatite Product Information Sheet, Rev B Click to download
2156 CHT Ceramic Hydroxyapatite &#151; A New Dimension in Chromatography of Biological Molecules, Rev C Click to download
6601 Enhancing the Flexibility of the NGC&#153; Chromatography System: Addition of a Refractive Index Detector for Wine Sample Analysis, Rev A Click to download
1801 Protein and Peptide Purification Applications, Rev B Click to download
6067 EDTA Complexometric Titration of Hydroxyapatite Column Effluent, Rev B Click to download
RP0033 A Ceramic Hydroxyapatite–Based Purification Platform &mdash; Simultaneous Removal of Leached Protein A, Aggregates, DNA, and Endotoxins from MAbs Click to download
2524 Purification of Horse IgG<sub>T</sub> Using Macro-Prep DEAE and CHT Ceramic Hydroxyapatite Type I Supports, Rev C Click to download
2774 Purification of Transgenic Antibody From Corn Seed Using UNOsphere S and CHT Ceramic Hydroxyapatite Supports, Rev A Click to download
5853 Chimeric IgG Monoclonal Antibody Purification: A Comparative Study Using CHT Ceramic Hydroxyapatite and CFT Ceramic Fluoroapatite Chromatographic Media, Rev A Click to download
6549 Mammalian Virus Purification Using Ceramic Hydroxyapatite, Rev A Click to download
6278 High-Throughput Calcium Analysis of Hydroxyapatite Column Effluent, Rev A Click to download
2731 Plasmid Purification Using CHT Ceramic Hydroxyapatite Support, Rev A Click to download
2780 Purification of Murine IgG<sub>1</sub> Using UNOsphere S and CHT Ceramic Hydroxyapatite Chromatography, Rev A Click to download
1986 Effect of pH on Gradient Elution of Proteins on Two Types of CHT Ceramic Hydroxyapatite, Rev B Click to download
2940 Removal of Aggregate from an IgG<sub>4</sub> Product Using CHT&trade; Ceramic Hydroxyapatite Resin, Rev B Click to download
2849 Protein A Removal From IgG on CHT Ceramic Hydroxyapatite Support, Rev A Click to download
2535 Purification of Recombinant Human alpha 1-Antitrypsin From Transgenic Milk Using CHT Ceramic Hydroxyapatite, Rev B Click to download
2575 Purification of a Calcium-Binding Protein Using CHT Ceramic Hydroxyapatite Click to download
2946 Plasmid DNA Adsorption Behavior on CHT Ceramic Hydroxyapatite Type II Support, Rev A Click to download
5831 Process-scale packing procedures for CHT ceramic hydroxyapatite in open column systems: Best practices, Rev A Click to download
5913 Separation of Fab and Fc Fragments from Monoclonal Antibody Papain Digest on Ceramic Hydroxyapatite and Ceramic Fluoroapatite, Rev A Click to download
6749 Mixed-Mode Chromatography for mAb S Aggregate Removal: Comparison of CHT™ Ceramic Hydroxyapatite, Capto adhere, and Capto adhere ImpRes, Ver A Click to download
 
 
MWHB6KKSY [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [64.237.54.6, 10.232.2.13] [seourl] = [/en-us/applications-technologies/multimodal-or-mixed-mode-chromatography] [x-amzn-trace-id] = [Root=1-5b4d7c5e-0401d3ac348d8a5cfe744120] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=MWHB6KKSY] [host] = [10.232.1.21:1776] [x-request-uri] = [/en-us/applications-technologies/multimodal-or-mixed-mode-chromatography] [connection] = [Keep-Alive] [cache-control] = [max-age=0] [accept-encoding] = [identity] [user-agent] = [Mozilla/5.0 (Windows NT 6.1; WOW64; rv:40.0) Gecko/20100101 Firefox/40.1] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/multimodal-or-mixed-mode-chromatography LSR MWHB6KKSY Multimodal or Mixed-Mode Chromatography Multimodal or Mixed-Mode Chromatography /webroot/web/html/lsr/solutions/technologies/chromatography <p>Multimodal or mixed-mode protein chromatography is based on media supports that have been functionalized with ligands capable of multiple modes of interaction: ion exchange, hydroxyapatite, affinity, size exclusion, and hydrophobic interactions. The ability to combine these separation methods can enhance selectivity in a protein purification process. This section provides an overview of multimodal chromatography, discusses some general considerations for mixed-mode chromatography, and describes hydrophobic ion-exchange and mixed-mode media and ligands.</p> <p>To find out whether multimodal chromatography is the most suitable approach for your application and to learn about other chromatographic methods that may be applicable for your purification needs, visit our <a href="/evportal/destination/solutions?catID=LUSMIS7OP">Chromatography</a> page.</p> <p><strong> Related Topics:</strong> <a href="/en-us/applications-technologies/liquid-chromatography/size-exclusion-chromatography">Size Exclusion Chromatography</a>, <a href="/en-us/applications-technologies/liquid-chromatography-principles/ion-exchange-chromatography">Ion Exchange Chromatography</a>, <a href="/evportal/destination/solutions?catID=MWHB53MNI">Hydrophobic Interaction Chromatography</a>, <a href="http://qaevn.bio-rad.com/en-us/applications-technologies/liquid-chromatography/affinity-chromatography">Affinity Chromatography</a>, <a href="/evportal/destination/solutions?catID=MWHBAYLPT">Low Pressure Chromatography Systems</a> and <a href="/evportal/destination/solutions?catID=MWHBD3HYP">Medium Pressure Chromatography Systems</a>.</p> General Considerations <p>Unlike affinity chromatography where a specific site on the protein is targeted, with mixed-mode ligands there is no known specificity. Accordingly, screening mixed-mode media becomes a search for sites on the target protein that will provide useful affinity and selectivity.</p> <p>Mixed-mode chromatography interactions are not independent of one another. For example, when using a mixed-mode ligand containing both hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds, however, increasing salt concentration will promote hydrophobic interactions.</p> <p>Mixed-mode media effectively combine complementary chromatography methods within a single media and can reduce the total number of column steps needed in a purification process. Because these mixed-mode elements are present in a single ligand, it contributes affinity-like binding and selectivity.</p> <p>Binding and elution are controlled and optimized by the parameters relevant to each mode &mdash; salt for hydrophobic interactions and ionic strength for ionic interactions.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Mixed-Mode Media <p>There are a number of commercially available mixed-mode media combining different chromatographic elements:</p> <ul> <li>Hydroxyapatite (CHT/CFT/Bio-Gel HT/HTP) &mdash; electrostatic and calcium coordination complexes</li> <li>Hydrophobic ion exchange ligands</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Hydroxyapatite Chromatography <p>Hydroxyapatite, Ca<sub>10</sub>(PO<sub>4</sub>)<sub>6</sub>(OH)<sub>2</sub>, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of &ndash;OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques.</p> <p>Applications of hydroxyapatite chromatography include the purification of:</p> <ul> <li>Different subclasses of monoclonal and polyclonal antibodies</li> <li>Antibodies that differ in light chain composition</li> <li>Antibody fragments</li> <li>Recombinant proteins </li> <li>Viral particles</li> <li>Vaccines</li> <li>Isozymes</li> <li>Supercoiled DNA from linear duplexes</li> <li>Single-stranded from double-stranded DNA</li> </ul> <p>CHT&trade; ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a nanocrystalline to a ceramic form. The ceramic material retains the unique separation properties of crystalline hydroxyapatite, and lot-to-lot control assures reproducibility in large-scale production columns. Unlike most other chromatography adsorbents, CHT is both the ligand and the support matrix. Separation protocols originally developed on crystalline hydroxyapatite can often be transferred directly to the ceramic material with only minor modifications.</p> <p>Two types of CHT ceramic hydroxyapatite, <a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=34cbb522-888f-43c7-aaf5-a61cfada935e">Type I</a> and <a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=a642d16e-5ca8-4981-acf2-a57271984632">Type II</a>, are available in three particle sizes, 20, 40, and 80 &mu;m. Although both types have elution characteristics similar to crystalline hydroxyapatite, there are also some important differences. CHT Type I has a higher protein binding capacity and better capacity for acidic proteins. CHT Type II has a lower protein binding capacity but has better resolution of nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/lab_scale_chromatography/chromatography/technology_detail/chromt15_img1.jpg" alt="" width="352" height="205" /></p> <p class="caption" style="width: 352px;"><strong>Protein binding to hydroxyapatite.</strong> <strong>A</strong> is a basic protein. <strong>B</strong> is an acidic protein. Double parentheses indicate repulsion. Dotted lines indicate ionic bonds. Triangular linkages indicate coordination bonds.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Hydrophobic Ion-Exchange Ligands <p>These ligands incorporate hydrophobic and ionic elements. For ion exchange binding and elution, parameters such as salt, pH, ionic strength, and buffer apply. With hydrophobic interactions, salt type, concentration, and additives apply. As mentioned above, it is important to remember that binding and elution factors are not independent and can work counter to one another. For example, increasing the ionic strength for ion-exchange elution will drive hydrophobic binding. Due to the dependency of these interactions, buffer conditions must be optimized for binding, washing, and elution to determine the optimal balance for a highly selective purification scheme.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Mixed-Mode Ligands <ul> <li><strong>Hydrophobic, Anionic Ligand with Hydrogen Bonding</strong> &mdash; this ligand features hydrogen bonding, a quaternary amine, and a phenyl group</li> <li><strong>Mixed-Mode Cationic Ligand with Hydrophobic Binding</strong> &mdash; this ligand contains a secondary amine and is cationic over a wide pH range, therefore, it behaves as both a hydrophobic interaction resin and an anionic exchange media. At low ionic strength, it can bind acidic proteins as well as proteins with moderately high isoelectric points. However, hydrophobic interactions predominate, as binding capacity increases with temperature and salt</li> <li><strong>Mixed-Mode pH-Controllable Sorbents</strong> &mdash; this ligand contains a 4-mercaptoethylpyridine (MEP) ligand. The pyridine ring is uncharged at neutral and basic pH. As the pH decreases, the pyridine nitrogen becomes positively charged, turning the resin into a mixed-mode media. MEP becomes a pH-controlled mixed-mode ligand, a property that has been termed "hydrophobic charge induction chromatography" (Burton and Harding, 1998)</li> </ul> <p><img style="float: left; margin-right:10px;" src="/webroot/web/images/lsr/solutions/technologies/lab_scale_chromatography/chromatography/technology_detail/chromt15_img2.jpg" alt="" width="263" height="229" /></p> <p class="caption" style="margin-left:20px; margin-top:85px"><strong>Example of mixed-mode ligands. A</strong> is a hydrophobic, anionic ligand with hydrogen bonding. <strong>B</strong> is a mixed-mode pH-controllable sorbent. <strong>C</strong> is a mixed-mode cationic ligand with hydrophobic binding.</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <div class="top"><a href="#helptop">Back to Top</a></div> Mixed Mode Chromatography Media Selection Guide <table class="pd_table" border="0"> <tbody> <tr> <td class="bordertop" colspan="2" valign="top">&nbsp;</td> <td class="bordertop borderbttm" colspan="3" align="center" valign="top"><strong>Suitability**</strong></td> <td class="bordertop" valign="top">&nbsp;</td> </tr> <tr> <td valign="bottom"><strong>Media Type</strong></td> <td valign="bottom"><strong>Packaging<br /> Format*</strong></td> <td valign="bottom"><strong>Analytical<br /> Scale</strong></td> <td valign="bottom"><strong>Pilot/<br />Preparative<br />Scale</strong></td> <td valign="bottom"><strong>Process<br /> Scale</strong></td> <td valign="bottom"><strong>Application</strong></td> </tr> <tr> <td class="bordertop" colspan="6" valign="top"><strong>Hydroxyapatite and Fluoroapatite</strong></td> </tr> <tr class="pd_colorbackground"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=7b9e4566-4a07-482a-8082-62dfcb1dd0b1">CHT&trade;<br />Type I</a></td> <td valign="top">B, C, MPC</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">Antibody purification (higher capacity than Type II); virus purification/removal; DNA purification/removal; aggregate and endotoxin removal; Fab purification</td> </tr> <tr class="pd_table pd_gridlines"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=5ded4ec5-ff5a-47b7-bf10-07253005c83f">CHT<br />Type II</a></td> <td valign="top">B, C</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">Antibody purification; removal of albumin from feedstream; Fab purification</td> </tr> <tr class="pd_colorbackground"> <td valign="top">CFT&trade;<br />Types I and II</td> <td valign="top">B, C</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">++++</td> <td valign="top">Similar properties to CHT but exhibits greater stability in the lower pH range (5.5); suitable for Fab purification</td> </tr> <tr class="pd_table pd_gridlines"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=ea60717e-e888-4b44-ac03-61600278c77c">Bio-Gel<sup>&reg;</sup> HT</a></td> <td valign="top">B</td> <td valign="top">++++</td> <td valign="top">+++</td> <td valign="top">&nbsp;</td> <td valign="top">Purification of proteins, nucleic acids and other biomolecules; crystalline hydroxyapatite not as mechanically stable as CHT (ceramic hydroxyapatite)</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><a href="http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=57f66f33-143c-402c-bde8-8cbd9ceebdb4">Bio-Gel<sup>&reg;</sup> HTP</a></td> <td valign="top">B</td> <td valign="top">++++</td> <td valign="top">+++</td> <td valign="top">&nbsp;</td> <td valign="top">Similar to Bio-Gel HT but in powder form</td> </tr> <tr class="pd_table pd_gridlines"> <td valign="top">DNA grade Bio-Gel<sup>&reg;</sup> HTP</td> <td valign="top">B</td> <td valign="top">++++</td> <td valign="top">+++</td> <td valign="top">&nbsp;</td> <td valign="top">Similar to Bio-Gel HTP with smaller particle size; selectivity for dsDNA; separation of ss- and dsDNA</td> </tr> </tbody> </table> <p class="caption">* B, bottle; C, cartridge (1 ml or 5 ml); GC, gravity column; SC, spin column; HPLC, high-pressure column; MPC, medium-pressure column.</p> <p class="caption">** +, low suitability; ++, moderate suitability; +++, suitable; ++++, high suitability.</p> <div class="top"><a href="#helptop">Back to Top</a></div> References <p>Burton SC and Harding DR (1998). Hydrophobic charge induction chromatography: Salt independent protein adsorption and facile elution with aqueous buffers. J Chromatogr A 814, 71&ndash;81.</p> <div class="top"><a href="#helptop">Back to Top</a></div> 5709 /templatedata/internet/documentation/data/LSR/Literature/5709_0910141009835.xml 6086 /templatedata/internet/documentation/data/LSR/Literature/6086.xml 5667 /templatedata/internet/documentation/data/LSR/Literature/5667_0910141009863.xml 2156 /templatedata/internet/documentation/data/LSR/Literature/2156_0910140920541.xml 6601 /templatedata/internet/documentation/data/LSR/Literature/6601_1411507329.xml 1801 /templatedata/internet/documentation/data/LSR/Literature/1801_0910140920099.xml 6067 /templatedata/internet/documentation/data/LSR/Literature/6067.xml RP0033 /templatedata/internet/documentation/data/PSD/Literature/RP0033.xml 2524 /templatedata/internet/documentation/data/LSR/Literature/2524_0910140920029.xml 2774 /templatedata/internet/documentation/data/LSR/Literature/2774_0910140920158.xml 5853 /templatedata/internet/documentation/data/LSR/Literature/5853_0910141008991.xml 6549 /templatedata/internet/documentation/data/LSR/Literature/6549_1411507790.xml 6278 /templatedata/internet/documentation/data/PSD/Literature/6278.xml 2731 /templatedata/internet/documentation/data/LSR/Literature/2731_0910140920376.xml 2780 /templatedata/internet/documentation/data/LSR/Literature/2780_0910140919395.xml 1986 /templatedata/internet/documentation/data/LSR/Literature/1986_0910140920404.xml 2940 /templatedata/internet/documentation/data/LSR/Literature/2940_0910140920460.xml 2849 /templatedata/internet/documentation/data/LSR/Literature/2849_0910140919369.xml 2535 /templatedata/internet/documentation/data/LSR/Literature/2535_0910140920431.xml 2575 /templatedata/internet/documentation/data/LSR/Literature/2575_0910140920488.xml 2946 /templatedata/internet/documentation/data/LSR/Literature/2946_0910140920515.xml 5831 /templatedata/internet/documentation/data/LSR/Literature/5831_0910141009806.xml 5825 /templatedata/internet/documentation/data/LSR/Literature/5825_0910141009892.xml 5913 /templatedata/internet/documentation/data/PSD/Literature/5913_1256598170.xml 6749 /templatedata/internet/documentation/data/LSR/Literature/6749_1450213106.xml Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/Deionizing Analytical Grade Resins ->MT::803a59da-012b-4afb-b0dc-e64e1d5e7d66##Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/CHT Ceramic Hydroxyapatite Type I Resin ->MT::34cbb522-888f-43c7-aaf5-a61cfada935e##Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/CHT Ceramic Hydroxyapatite Type II Resin ->MT::a642d16e-5ca8-4981-acf2-a57271984632## Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/Bio-Gel Hydroxyapatite HT and HTP Media ->MT::1e754042-4d9d-4487-ba7b-dc67dec9f8fc##Life Science Research/Products/Chromatography/Chromatography Media/Multimodal Chromatography Media/Fluoroapatite Media ->MT::f6456568-f543-48d2-a210-b727ab3137e8## Life Science Research/Solutions/Technologies/Protein Electrophoresis/Protein Sample Preparation ->MTS::LUSP108WI##Life Science Research/Solutions/Applications/Automated Electrophoresis and Analysis ->MTS::LUSQXB15##Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Applications/Protein Interaction Analysis ->MTS::LUSLTJE8Z##Life Science Research/Solutions/Technologies/Surface Plasmon Resonance ->MTS::LUSM664EH## Karen Moss Multimodal or Mixed-Mode Chromatography 11/20/13 01:26 PM 11/20/23 01:40 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/Chromatography N 0
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