Antibody Purification

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en-us MA7DUU15 Monoclonal Antibody and Fragment Discovery Antibody Purification /webroot/web/html/lsr/solutions/applications/protein_purification <p>In recent years, drug discovery researchers have expanded their portfolio of products from small-molecule drugs to large biomolecules such as recombinant proteins or monoclonal antibodies developed for diagnostic or therapeutic uses. The production of these important biomolecules often requires many chromatographic steps to obtain pure products and ensure that no contaminants are present that might cause adverse effects such as allergic reactions or even death.</p> <p><strong>Related Topics: </strong><a href="/evportal/destination/solutions?catID=MA7DZ94VY">Histidine-Tagged Recombinant Protein Purification and On-Column Refolding</a>, <a href="http://www.bio-rad.com/en-us/applications-technologies/liquid-chromatography-principles/affinity-chromatography">Affinity Chromatography</a>, and <a href="/en-us/applications-technologies/protein-purification-isolation">Protein Purification and Isolation</a>.</p> <br /> Monoclonal Antibody Discovery and Development <p>The amount of purified protein required differs depending on the research and development phase in which the biomolecule of interest is being evaluated. For example, in the early discovery phase, smaller amounts of multiple antibody constructs, each with different binding characteristics, are required for testing and analysis.</p> <p>Methods such as <a href="/en-us/applications-technologies/label-free-biomolecular-interactions">surface plasmon resonance</a> are used to determine the kinetic parameters for the binding of these antibodies with their targets and to allow for the identification of antibodies that have the desired binding characteristics. These antibodies are then further investigated in the preclinical phase. Ultimately, clinical trials are performed to determine the effectiveness of experimental drugs in humans.</p> <p>Once an effective antibody has been identified, the last phase of the drug-production workflow requires large amounts of purified product. At the large scale, similar separation techniques are utilized but with a much higher throughput.</p> <p>There are many different methods for antibody production; two of the most popular methods are the creation of antibodies and antibody fragments from mouse hybridoma cell lines, and phage display. Phage display is a common method for the production of antibody fragments (Fabs), so named because these fragments contain only an antigen-binding region (see Figure 1).</p> <div style="float: left; width: 147px; margin-right: 20px; margin-bottom: 20px;"><img src="/webroot/web/images/lsr/solutions/applications/protein_purification_and_isolation/application_detail/schematic-structure-of-an-antibody.jpg" alt="Schematic structure of an antibody" width="147" height="144" /><br /> <span class="caption"><strong>Fig. 1. Schematic structure of an antibody.</strong> Fab, antigen binding region; Fc, crystallizable region.</span></div> <p>Phage display is widely used by researchers in the development of novel therapies or the identification of novel targets for therapies because millions of Fabs can be produced in a single experiment. These Fabs are subjected to an initial screening to identify those with highest affinity for their intended targets. Successfully selected Fabs are often converted into full-length antibodies for possible therapeutic use; however, successful translation into clinical practice remains rare.</p> <p>Both methods rely on chromatography to isolate and purify antibodies and antibody fragments (Fabs) from complex media, such as cultured mouse cell lines or bacterial supernatants. Because these media contain highly complex protein mixtures, extensive multistep chromatography is often required to produce purified antibody products.</p> <p>&nbsp;</p> <div class="top"><a href="#helptop">Back to Top</a></div> Antibody Purification Methods <p>Monoclonal antibodies are typically purified from crude samples derived from either culture media or ascites taken from a host animal. These crude samples contain many contaminants such as growth factors, hormones, DNA, endotoxins, and host cell proteins.</p> <p>A typical workflow for monoclonal antibody purification involves initial centrifugation and filtration steps prior to chromatography. Many chromatographic methods can be employed in antibody purification.</p> <p>A popular method utilizes <a href="http://www.bio-rad.com/en-us/applications-technologies/liquid-chromatography-principles/affinity-chromatography">affinity chromatography</a>. Here, the crude sample is passed through a column filled with a resinous stationary phase containing protein A, which captures the antibody because protein A has a high specificity for the Fc (fragment, crystallizable) regions of antibodies (see Figure 1).</p> <p>Although affinity chromatography is a simple and quick approach, it has the drawback of being expensive compared to other methods. Much of this expense is due to the shorter lifetime of protein A resin compared with other stationary phases.</p> <p>Additionally, affinity chromatography is only a part of a multistep approach wherein the crude sample is first passed through an affinity column and subsequently polished using <a href="/en-us/applications-technologies/liquid-chromatography-principles/ion-exchange-chromatography">ion exchange chromatography (IEX)</a>.</p> <p><a href="/en-us/applications-technologies/cation-exchange-chromatography">Cation exchange chromatography</a> is a popular method for cleaning up an isolated monoclonal antibody. In this step, the antibody binds the solid phase, and contaminants are allowed to flow through or wash off the protein.</p> <p>The complementary method, <a href="/en-us/applications-technologies/anion-exchange-chromatography">anion exchange chromatography</a>, may also be employed, in which case contaminants are captured on the anion exchange column, while the antibody flows through. Two ion-exchange chromatography steps are often needed for the complete removal of cell-related contaminants.</p> <p><a href="/en-us/applications-technologies/liquid-chromatography-principles/size-exclusion-chromatography">Size-exclusion chromatography (SEC)</a> is a less popular final step for removing residual proteins and contaminants; this method often employs a size-based filter rather than a column due to volume constraints.</p> <p>Tagged affinity chromatography is another frequently employed method. In this process, a recombinant antibody is produced as a fusion protein containing a terminal affinity tag such as a polyhistidine tag. The chromatography column contains media functionalized with a molecule that binds the tag with high affinity; for example, transition metal compounds, especially nickel compounds such as tris-NTA, bind histidine residues tightly yet reversibly. This method enables antibody capture and purification in one step in a process called <a href="/en-us/applications-technologies/types-chromatography#imac">immobilized metal ion affinity chromatography (IMAC)</a>. However, this approach yields an antibody with an attached tag, which may interfere with the binding of the antibody to its target antigen. This tag must therefore be removed, adding downstream processing to the workflow.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Removal of Viral Contaminants from Antibody Preparations <div style="float: left; width: 118px; height: 500px; margin-right: 20px; margin-bottom: 20px;"><img src="/webroot/web/images/lsr/solutions/applications/protein_purification_and_isolation/application_detail/chromatography-process-flow-diagram-for-antibody-purification-and-virus-removal.jpg" alt="Flow diagram for antibody purification and virus removal" width="98" height="467" /><br /><span class="caption"><strong>Fig. 2. Chromatography process flow diagram for antibody purification and virus removal.</strong> </span></div> <p>Therapeutic antibody production on the process scale demands tens of grams of highly pure product. This product must be free from contaminants such as viruses that may cause adverse events in patients. The production of virus-free cetuximab provides an illustrative example.</p> <p>Cetuximab is a chimeric monoclonal antibody with a high specificity for human epidermal growth factor receptor. This antibody is currently used to treat patients with metastatic cancers.</p> <p>Several different methods have been tested to produce a virus-free antibody product; one such method using a two-column chromatographic procedure is described below.</p> <p>In this scaled-down example, the antibody is grown in serum-free production media and spiked with the Moloney ecotropic murine leukemia virus (MuLV). This model envelope retrovirus is approximately 80&ndash;110 nm in diameter.</p> <p>The purification process involves a two-column chromatographic procedure utilizing strong anion and strong cation exchange chromatography (shown to the side). A known titer of the MuLV, here 1.9 &times; 10<sup>6</sup> plaque-forming units (PFU) per ml, is spiked into the antibody preparation and loaded onto an anionic column.</p> <p>This column strongly binds contaminants such as DNA and RNA, and the effluent contains purified protein and MuLV at a lower concentration than in the original solution (5.75 &times; 10<sup>5</sup> PFU/ml).</p> <p>The effluent is then loaded onto the cation exchange column, where the antibody binds to the column. The column is washed free of contaminants, and the antibody is then eluted by &nbsp;applying a pH gradient.</p> <p><a href="/webroot/web/pdf/lsr/literature/Bulletin_1985.pdf" target="_blank">This two-column method successfully yields purified antibody and reduces viral load eightfold</a>, thus meeting the U.S. Food and Drug Administration safety requirements.</p> <div class="top"><a href="#helptop">Back to Top</a><br /><br /><br /><br /><br /></div> 6966 /templatedata/internet/documentation/data/LSR/Literature/6966_1502486227.xml Development of a Non–Affinity Based Purification Platform for Neutral/Basic IgMs 6966 /webroot/web/pdf/lsr/literature/Bulletin_6966.pdf Literature PDF Articles_and_Whitepapers Development of a Non–Affinity Based Purification Platform for Neutral/Basic IgMs No Development of a Non–Affinity Based Purification Platform for Neutral/Basic IgMs 6966 6966, nuvia s, cht, ceramic, hydroxyapatite, foresight, ngc, antibody, protein, purification, igm, chromatography, cation, exchange, cex, mixed-mode, mixed mode, affinity, capture, polish, mini-protean, miniprotean, mini protean, chemidoc, 7324720, 7324736, 7880011, 4568093, 17001402 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 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 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 2026 /templatedata/internet/documentation/data/LSR/Literature/2026_0910140919636.xml Evaluation of Different Approaches for the Chromatographic Purification of Monoclonal Antibodies 2026 /webroot/web/pdf/lsr/literature/Bulletin_2026.pdf Literature PDF Application_Notes /webroot/web/images/general/icons/icon_pdf.gif No Evaluation of Different Approaches for the Chromatographic Purification of Monoclonal Antibodies 2026 bulletin 2026, lit2026, chromatography, column, columns, medium, media, resin, resins, protein, purification, monoclonal, antibody, affinity, ion, exchange, iex, anion, cation, anex, catex, cex 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 1917 /templatedata/internet/documentation/data/LSR/Literature/1917_0910140921903.xml Purification of a Human Monoclonal IgM Antibody From Bioreactor Supernatant Using a Combination of Cation and Anion Exchange 1917 /webroot/web/pdf/lsr/literature/Bulletin_1917.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Purification of a Human Monoclonal IgM Antibody From Bioreactor Supernatant Using a Combination of Cation and Anion Exchange 1917 lit1917, bulletin 1917, chromatography, column, columns, medium, media, resin, resins, protein, purification, ion, exchange, iex, anion, cation, anex, catex, cex, macro-prep, macroprep, macro prep, scale-up, monoclonal, antibody, antibodies, igg, igm, process scale, scale-up 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 1985 /templatedata/internet/documentation/data/LSR/Literature/1985_0910140919998.xml Clearance of Murine Leukemia Virus From a Chimeric Monoclonal Antibody Using Ion Exchange Chromatography, Rev B. 1985 /webroot/web/pdf/lsr/literature/Bulletin_1985.pdf Literature PDF Articles_and_Whitepapers /webroot/web/images/general/icons/icon_pdf.gif No A Poster Presentation 1985 bulletin 1985, lit1985, chromatography, column, columns, medium, media, resin, resins, protein, purification, monoclonal, antibody, virus, ion, exchange, iex, anion, cation, anex, catex, cex, macro-prep 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 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 6694 /templatedata/internet/documentation/data/LSR/Literature/6694_1428946376.xml Advantages of Multidimensional (Multi-D) Chromatography Using the NGC™ Chromatography System over Traditional Sequential Chromatography, Ver B 6694 /webroot/web/pdf/lsr/literature/Bulletin_6694.pdf Literature PDF Application_Notes Advantages of Multidimensional (Multi-D) Chromatography Using the NGC™ Chromatography System over Traditional Sequential Chromatography, Ver B No Advantages of Multidimensional (Multi-D) Chromatography Using the NGC™ Chromatography System over Traditional Sequential Chromatography, Ver B 6694 6694, ngc, chromatography, chromatography system, discover pro, chromlab, multidimensional chromatography, multidimensional, multi-D, multiD, tandem chromatography, tandem, sequential chromatography, automated purification, protein purification, antibody purification, biologics, aggregation, protein a, affinity, size exclusion chromatography, sec, desalting, bio-scale mini, bioscale mini, unosphere supra, enrich sec 650, bio-gel p-6, biogel p-6, biogel p6, bio-gel p6, 788-0012, 7880012, 788-6000, 7886000 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 6781 /templatedata/internet/documentation/data/LSR/Literature/6781_1465432237.xml Life Science Research/Products/Chromatography/Chromatography Media/Size Exclusion Chromatography Media/Bio-Gel A Agarose Gel ->MT::7d922386-85be-4295-a797-e574d7b5f4f6##Life Science Research/Products/Chromatography/Chromatography Media/Cation Exchange Chromatography Media/UNOsphere Cation Exchange Media ->MT::475eb93b-673a-40f8-a055-7a05d41be4b3##Life Science Research/Products/Chromatography/Chromatography Media/Affinity Chromatography Media/Profinity IMAC Resins ->MT::e54ab03c-d281-4e6b-aa0d-193b7737626d##Life Science Research/Products/Chromatography/Chromatography Media/Anion Exchange Chromatography Media/UNOsphere Anion Exchange Media ->MT::36286010-253e-4553-b13d-8b5917f8e93a## Karen Moss 09/17/12 10:21 AM 09/19/22 12:57 AM 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/Applications/Protein_Purification_and_Isolation N 0 Protein Purification and Isolation /en-us/applications-technologies/applications-technologies/antibody-purification?ID=M94H8Z15

In recent years, drug discovery researchers have expanded their portfolio of products from small-molecule drugs to large biomolecules such as recombinant proteins or monoclonal antibodies developed for diagnostic or therapeutic uses. The production of these important biomolecules often requires many chromatographic steps to obtain pure products and ensure that no contaminants are present that might cause adverse effects such as allergic reactions or even death.

Related Topics: Histidine-Tagged Recombinant Protein Purification and On-Column Refolding, Affinity Chromatography, and Protein Purification and Isolation.


 

Monoclonal Antibody Discovery and Development

The amount of purified protein required differs depending on the research and development phase in which the biomolecule of interest is being evaluated. For example, in the early discovery phase, smaller amounts of multiple antibody constructs, each with different binding characteristics, are required for testing and analysis.

Methods such as surface plasmon resonance are used to determine the kinetic parameters for the binding of these antibodies with their targets and to allow for the identification of antibodies that have the desired binding characteristics. These antibodies are then further investigated in the preclinical phase. Ultimately, clinical trials are performed to determine the effectiveness of experimental drugs in humans.

Once an effective antibody has been identified, the last phase of the drug-production workflow requires large amounts of purified product. At the large scale, similar separation techniques are utilized but with a much higher throughput.

There are many different methods for antibody production; two of the most popular methods are the creation of antibodies and antibody fragments from mouse hybridoma cell lines, and phage display. Phage display is a common method for the production of antibody fragments (Fabs), so named because these fragments contain only an antigen-binding region (see Figure 1).

Schematic structure of an antibody
Fig. 1. Schematic structure of an antibody. Fab, antigen binding region; Fc, crystallizable region.

Phage display is widely used by researchers in the development of novel therapies or the identification of novel targets for therapies because millions of Fabs can be produced in a single experiment. These Fabs are subjected to an initial screening to identify those with highest affinity for their intended targets. Successfully selected Fabs are often converted into full-length antibodies for possible therapeutic use; however, successful translation into clinical practice remains rare.

Both methods rely on chromatography to isolate and purify antibodies and antibody fragments (Fabs) from complex media, such as cultured mouse cell lines or bacterial supernatants. Because these media contain highly complex protein mixtures, extensive multistep chromatography is often required to produce purified antibody products.

 

 

Antibody Purification Methods

Monoclonal antibodies are typically purified from crude samples derived from either culture media or ascites taken from a host animal. These crude samples contain many contaminants such as growth factors, hormones, DNA, endotoxins, and host cell proteins.

A typical workflow for monoclonal antibody purification involves initial centrifugation and filtration steps prior to chromatography. Many chromatographic methods can be employed in antibody purification.

A popular method utilizes affinity chromatography. Here, the crude sample is passed through a column filled with a resinous stationary phase containing protein A, which captures the antibody because protein A has a high specificity for the Fc (fragment, crystallizable) regions of antibodies (see Figure 1).

Although affinity chromatography is a simple and quick approach, it has the drawback of being expensive compared to other methods. Much of this expense is due to the shorter lifetime of protein A resin compared with other stationary phases.

Additionally, affinity chromatography is only a part of a multistep approach wherein the crude sample is first passed through an affinity column and subsequently polished using ion exchange chromatography (IEX).

Cation exchange chromatography is a popular method for cleaning up an isolated monoclonal antibody. In this step, the antibody binds the solid phase, and contaminants are allowed to flow through or wash off the protein.

The complementary method, anion exchange chromatography, may also be employed, in which case contaminants are captured on the anion exchange column, while the antibody flows through. Two ion-exchange chromatography steps are often needed for the complete removal of cell-related contaminants.

Size-exclusion chromatography (SEC) is a less popular final step for removing residual proteins and contaminants; this method often employs a size-based filter rather than a column due to volume constraints.

Tagged affinity chromatography is another frequently employed method. In this process, a recombinant antibody is produced as a fusion protein containing a terminal affinity tag such as a polyhistidine tag. The chromatography column contains media functionalized with a molecule that binds the tag with high affinity; for example, transition metal compounds, especially nickel compounds such as tris-NTA, bind histidine residues tightly yet reversibly. This method enables antibody capture and purification in one step in a process called immobilized metal ion affinity chromatography (IMAC). However, this approach yields an antibody with an attached tag, which may interfere with the binding of the antibody to its target antigen. This tag must therefore be removed, adding downstream processing to the workflow.

 

Removal of Viral Contaminants from Antibody Preparations

Flow diagram for antibody purification and virus removal
Fig. 2. Chromatography process flow diagram for antibody purification and virus removal.

Therapeutic antibody production on the process scale demands tens of grams of highly pure product. This product must be free from contaminants such as viruses that may cause adverse events in patients. The production of virus-free cetuximab provides an illustrative example.

Cetuximab is a chimeric monoclonal antibody with a high specificity for human epidermal growth factor receptor. This antibody is currently used to treat patients with metastatic cancers.

Several different methods have been tested to produce a virus-free antibody product; one such method using a two-column chromatographic procedure is described below.

In this scaled-down example, the antibody is grown in serum-free production media and spiked with the Moloney ecotropic murine leukemia virus (MuLV). This model envelope retrovirus is approximately 80–110 nm in diameter.

The purification process involves a two-column chromatographic procedure utilizing strong anion and strong cation exchange chromatography (shown to the side). A known titer of the MuLV, here 1.9 × 106 plaque-forming units (PFU) per ml, is spiked into the antibody preparation and loaded onto an anionic column.

This column strongly binds contaminants such as DNA and RNA, and the effluent contains purified protein and MuLV at a lower concentration than in the original solution (5.75 × 105 PFU/ml).

The effluent is then loaded onto the cation exchange column, where the antibody binds to the column. The column is washed free of contaminants, and the antibody is then eluted by  applying a pH gradient.

This two-column method successfully yields purified antibody and reduces viral load eightfold, thus meeting the U.S. Food and Drug Administration safety requirements.

 

Related Content

 
Literature
Number Description Download
6966 Development of a Non–Affinity Based Purification Platform for Neutral/Basic IgMs 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
RP0033 A Ceramic Hydroxyapatite–Based Purification Platform &mdash; Simultaneous Removal of Leached Protein A, Aggregates, DNA, and Endotoxins from MAbs Click to download
2774 Purification of Transgenic Antibody From Corn Seed Using UNOsphere S and CHT Ceramic Hydroxyapatite Supports, Rev A Click to download
2026 Evaluation of Different Approaches for the Chromatographic Purification of Monoclonal Antibodies 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
1917 Purification of a Human Monoclonal IgM Antibody From Bioreactor Supernatant Using a Combination of Cation and Anion Exchange Click to download
2780 Purification of Murine IgG<sub>1</sub> Using UNOsphere S and CHT Ceramic Hydroxyapatite Chromatography, Rev A Click to download
1985 Clearance of Murine Leukemia Virus From a Chimeric Monoclonal Antibody Using Ion Exchange Chromatography, 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
5913 Separation of Fab and Fc Fragments from Monoclonal Antibody Papain Digest on Ceramic Hydroxyapatite and Ceramic Fluoroapatite, Rev A Click to download
6694 Advantages of Multidimensional (Multi-D) Chromatography Using the NGC™ Chromatography System over Traditional Sequential Chromatography, Ver B 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
 
 
MA7DUU15 [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [78.128.8.93, 10.232.18.193] [seourl] = [/en-us/applications-technologies/antibody-purification] [x-amzn-trace-id] = [Root=1-5b7931ed-71defea03ab1b3006515c4f8] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=MA7DUU15] [host] = [10.232.0.21:1776] [x-request-uri] = [/en-us/applications-technologies/antibody-purification] [connection] = [Keep-Alive] [cache-control] = [max-age=0] [accept-encoding] = [identity] [user-agent] = [Mozilla/5.0 (compatible; Googlebot/2.1; +http://www.google.com/bot.html)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/antibody-purification LSR MA7DUU15 Monoclonal Antibody and Fragment Discovery Antibody Purification /webroot/web/html/lsr/solutions/applications/protein_purification <p>In recent years, drug discovery researchers have expanded their portfolio of products from small-molecule drugs to large biomolecules such as recombinant proteins or monoclonal antibodies developed for diagnostic or therapeutic uses. The production of these important biomolecules often requires many chromatographic steps to obtain pure products and ensure that no contaminants are present that might cause adverse effects such as allergic reactions or even death.</p> <p><strong>Related Topics: </strong><a href="/evportal/destination/solutions?catID=MA7DZ94VY">Histidine-Tagged Recombinant Protein Purification and On-Column Refolding</a>, <a href="http://www.bio-rad.com/en-us/applications-technologies/liquid-chromatography-principles/affinity-chromatography">Affinity Chromatography</a>, and <a href="/en-us/applications-technologies/protein-purification-isolation">Protein Purification and Isolation</a>.</p> <br /> Monoclonal Antibody Discovery and Development <p>The amount of purified protein required differs depending on the research and development phase in which the biomolecule of interest is being evaluated. For example, in the early discovery phase, smaller amounts of multiple antibody constructs, each with different binding characteristics, are required for testing and analysis.</p> <p>Methods such as <a href="/en-us/applications-technologies/label-free-biomolecular-interactions">surface plasmon resonance</a> are used to determine the kinetic parameters for the binding of these antibodies with their targets and to allow for the identification of antibodies that have the desired binding characteristics. These antibodies are then further investigated in the preclinical phase. Ultimately, clinical trials are performed to determine the effectiveness of experimental drugs in humans.</p> <p>Once an effective antibody has been identified, the last phase of the drug-production workflow requires large amounts of purified product. At the large scale, similar separation techniques are utilized but with a much higher throughput.</p> <p>There are many different methods for antibody production; two of the most popular methods are the creation of antibodies and antibody fragments from mouse hybridoma cell lines, and phage display. Phage display is a common method for the production of antibody fragments (Fabs), so named because these fragments contain only an antigen-binding region (see Figure 1).</p> <div style="float: left; width: 147px; margin-right: 20px; margin-bottom: 20px;"><img src="/webroot/web/images/lsr/solutions/applications/protein_purification_and_isolation/application_detail/schematic-structure-of-an-antibody.jpg" alt="Schematic structure of an antibody" width="147" height="144" /><br /> <span class="caption"><strong>Fig. 1. Schematic structure of an antibody.</strong> Fab, antigen binding region; Fc, crystallizable region.</span></div> <p>Phage display is widely used by researchers in the development of novel therapies or the identification of novel targets for therapies because millions of Fabs can be produced in a single experiment. These Fabs are subjected to an initial screening to identify those with highest affinity for their intended targets. Successfully selected Fabs are often converted into full-length antibodies for possible therapeutic use; however, successful translation into clinical practice remains rare.</p> <p>Both methods rely on chromatography to isolate and purify antibodies and antibody fragments (Fabs) from complex media, such as cultured mouse cell lines or bacterial supernatants. Because these media contain highly complex protein mixtures, extensive multistep chromatography is often required to produce purified antibody products.</p> <p>&nbsp;</p> <div class="top"><a href="#helptop">Back to Top</a></div> Antibody Purification Methods <p>Monoclonal antibodies are typically purified from crude samples derived from either culture media or ascites taken from a host animal. These crude samples contain many contaminants such as growth factors, hormones, DNA, endotoxins, and host cell proteins.</p> <p>A typical workflow for monoclonal antibody purification involves initial centrifugation and filtration steps prior to chromatography. Many chromatographic methods can be employed in antibody purification.</p> <p>A popular method utilizes <a href="http://www.bio-rad.com/en-us/applications-technologies/liquid-chromatography-principles/affinity-chromatography">affinity chromatography</a>. Here, the crude sample is passed through a column filled with a resinous stationary phase containing protein A, which captures the antibody because protein A has a high specificity for the Fc (fragment, crystallizable) regions of antibodies (see Figure 1).</p> <p>Although affinity chromatography is a simple and quick approach, it has the drawback of being expensive compared to other methods. Much of this expense is due to the shorter lifetime of protein A resin compared with other stationary phases.</p> <p>Additionally, affinity chromatography is only a part of a multistep approach wherein the crude sample is first passed through an affinity column and subsequently polished using <a href="/en-us/applications-technologies/liquid-chromatography-principles/ion-exchange-chromatography">ion exchange chromatography (IEX)</a>.</p> <p><a href="/en-us/applications-technologies/cation-exchange-chromatography">Cation exchange chromatography</a> is a popular method for cleaning up an isolated monoclonal antibody. In this step, the antibody binds the solid phase, and contaminants are allowed to flow through or wash off the protein.</p> <p>The complementary method, <a href="/en-us/applications-technologies/anion-exchange-chromatography">anion exchange chromatography</a>, may also be employed, in which case contaminants are captured on the anion exchange column, while the antibody flows through. Two ion-exchange chromatography steps are often needed for the complete removal of cell-related contaminants.</p> <p><a href="/en-us/applications-technologies/liquid-chromatography-principles/size-exclusion-chromatography">Size-exclusion chromatography (SEC)</a> is a less popular final step for removing residual proteins and contaminants; this method often employs a size-based filter rather than a column due to volume constraints.</p> <p>Tagged affinity chromatography is another frequently employed method. In this process, a recombinant antibody is produced as a fusion protein containing a terminal affinity tag such as a polyhistidine tag. The chromatography column contains media functionalized with a molecule that binds the tag with high affinity; for example, transition metal compounds, especially nickel compounds such as tris-NTA, bind histidine residues tightly yet reversibly. This method enables antibody capture and purification in one step in a process called <a href="/en-us/applications-technologies/types-chromatography#imac">immobilized metal ion affinity chromatography (IMAC)</a>. However, this approach yields an antibody with an attached tag, which may interfere with the binding of the antibody to its target antigen. This tag must therefore be removed, adding downstream processing to the workflow.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Removal of Viral Contaminants from Antibody Preparations <div style="float: left; width: 118px; height: 500px; margin-right: 20px; margin-bottom: 20px;"><img src="/webroot/web/images/lsr/solutions/applications/protein_purification_and_isolation/application_detail/chromatography-process-flow-diagram-for-antibody-purification-and-virus-removal.jpg" alt="Flow diagram for antibody purification and virus removal" width="98" height="467" /><br /><span class="caption"><strong>Fig. 2. Chromatography process flow diagram for antibody purification and virus removal.</strong> </span></div> <p>Therapeutic antibody production on the process scale demands tens of grams of highly pure product. This product must be free from contaminants such as viruses that may cause adverse events in patients. The production of virus-free cetuximab provides an illustrative example.</p> <p>Cetuximab is a chimeric monoclonal antibody with a high specificity for human epidermal growth factor receptor. This antibody is currently used to treat patients with metastatic cancers.</p> <p>Several different methods have been tested to produce a virus-free antibody product; one such method using a two-column chromatographic procedure is described below.</p> <p>In this scaled-down example, the antibody is grown in serum-free production media and spiked with the Moloney ecotropic murine leukemia virus (MuLV). This model envelope retrovirus is approximately 80&ndash;110 nm in diameter.</p> <p>The purification process involves a two-column chromatographic procedure utilizing strong anion and strong cation exchange chromatography (shown to the side). A known titer of the MuLV, here 1.9 &times; 10<sup>6</sup> plaque-forming units (PFU) per ml, is spiked into the antibody preparation and loaded onto an anionic column.</p> <p>This column strongly binds contaminants such as DNA and RNA, and the effluent contains purified protein and MuLV at a lower concentration than in the original solution (5.75 &times; 10<sup>5</sup> PFU/ml).</p> <p>The effluent is then loaded onto the cation exchange column, where the antibody binds to the column. The column is washed free of contaminants, and the antibody is then eluted by &nbsp;applying a pH gradient.</p> <p><a href="/webroot/web/pdf/lsr/literature/Bulletin_1985.pdf" target="_blank">This two-column method successfully yields purified antibody and reduces viral load eightfold</a>, thus meeting the U.S. Food and Drug Administration safety requirements.</p> <div class="top"><a href="#helptop">Back to Top</a><br /><br /><br /><br /><br /></div> 6966 /templatedata/internet/documentation/data/LSR/Literature/6966_1502486227.xml 2524 /templatedata/internet/documentation/data/LSR/Literature/2524_0910140920029.xml RP0033 /templatedata/internet/documentation/data/PSD/Literature/RP0033.xml 2774 /templatedata/internet/documentation/data/LSR/Literature/2774_0910140920158.xml 2026 /templatedata/internet/documentation/data/LSR/Literature/2026_0910140919636.xml 5853 /templatedata/internet/documentation/data/LSR/Literature/5853_0910141008991.xml 1917 /templatedata/internet/documentation/data/LSR/Literature/1917_0910140921903.xml 2780 /templatedata/internet/documentation/data/LSR/Literature/2780_0910140919395.xml 1985 /templatedata/internet/documentation/data/LSR/Literature/1985_0910140919998.xml 2940 /templatedata/internet/documentation/data/LSR/Literature/2940_0910140920460.xml 2849 /templatedata/internet/documentation/data/LSR/Literature/2849_0910140919369.xml 5913 /templatedata/internet/documentation/data/PSD/Literature/5913_1256598170.xml 6694 /templatedata/internet/documentation/data/LSR/Literature/6694_1428946376.xml 6749 /templatedata/internet/documentation/data/LSR/Literature/6749_1450213106.xml 6781 /templatedata/internet/documentation/data/LSR/Literature/6781_1465432237.xml Life Science Research/Products/Chromatography/Chromatography Media/Size Exclusion Chromatography Media/Bio-Gel A Agarose Gel ->MT::7d922386-85be-4295-a797-e574d7b5f4f6##Life Science Research/Products/Chromatography/Chromatography Media/Cation Exchange Chromatography Media/UNOsphere Cation Exchange Media ->MT::475eb93b-673a-40f8-a055-7a05d41be4b3##Life Science Research/Products/Chromatography/Chromatography Media/Affinity Chromatography Media/Profinity IMAC Resins ->MT::e54ab03c-d281-4e6b-aa0d-193b7737626d##Life Science Research/Products/Chromatography/Chromatography Media/Anion Exchange Chromatography Media/UNOsphere Anion Exchange Media ->MT::36286010-253e-4553-b13d-8b5917f8e93a## Karen Moss 09/17/12 10:21 AM 09/19/22 12:57 AM 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/Applications/Protein_Purification_and_Isolation N 0
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