Protein A chromatography exploits the fact that murine IgG binds to Protein A Sepharose at pH>8 but does not bind at pH<3.0. Typically, the pH of the MAb-containing solution is adjusted to 8.5 and passed over a column of Protein A. With the MAb bound to the protein A, contaminants are washed from the column with a pH 8.5 buffer, Finally, the purified MAb is eluted by passing a pH 3.0 buffer over the column. (Profy, EP0282308A2)

The interaction between IgG and Protein A has been studied in detail and the interaction has been shown to primarily consist of hydrophobic interactions along with some hydrogen bonding and two salt bridges. The primary binding site for Protein A on the Fc region is at the juncture of Cgamma2 and Cgamma3 domains. Variations in affinity amongst IgGs of different species and subclasses for Protein A have been reported. Antibodies belonging to the same sublcass have greater than 95% homologus Fc regions and are expected to have similar binding affinity. Studies have revealed that a highly conserved histidyl residue is present in the center of the Protein A binding site of IgG which aligns facing a complimentary and similarly conserved histidine residue on Protein A itself. At alkaline or neutral pH, these residues are unchared and there are no restrictions on interfacial contact. At low pHs, the complementary histidine groups take on a positive charge resulting in electrostatic repulsion between the two proteins and a concomitant reduction in hydrophobic contact area between them. This electrostatic repulsion is strong enough to elute the antibody off the Protein A column. Elution pH is a critical parameter during Protein A process development. It has been observed that the eltuion pH can vary significantly even among antibodies which have homologous Fc region which is surprising based on conventioanl wisdom that the itneraction with Protein A is primarily dictated by the Fc region of the antibody. In addition to the classical binding stie, some immunoglobulins have been shown to have an “alternate binding” site for Protein A on their heavy chain variable domain. In particular, IgMs as well as some IgGs and IgAs that contain heavy chains from the human VH3 gene family have been shown to exhibit this behavior. Binding studies between Protein A and antibodies have shown that while all 5 domains of Sp1 (E, D, A, B, C) binding IgG via their Fc region, only domains D and E exhibit significant Fab bidning.  The Z domain, a functional analog of the B domain was shown to have negligible bidning to the antibody variable region.   (Ghose, Biotechnol Bioneg. 2005, 92(6), 665-73). See also Starovasnik, Protein Sci, 1999, 8(7), 1423-31) showing that immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc porition of the antibody.

Cell Culture Adjustment

Additiona of Benzoate:

Beigie (US 16/228291, published as US 2019/0233468) discloses that adjustment of a harvest to 0.5 M soidum benzoate at pH 7.2 or 0.5 M sodium sodium benzoate and pH 9 prior to protein A purificaiton leg to improved removal of PLBL2 and HCP impurities. 

Binding

The interaction of immobilized Protein A or Protein G with immunoglobulins is pH dependent. The binding capacity for Protein A is optimal at pH 8.9, whereas the binding capacity of Protein G is high over a broader pH range. “Protein A Antibody Purification Handbook” Pro-Chem Inc. 2005.  Protein A column typically bind antibodies at neutral pH and can be eluted at low pH (typically between pH 3 and 4). It has been shown that a highly conserved histidyl residue in the center of the protein A binding region of IgG faces a complementary histidyl residue on protein A. Those residues take on a positive charge at low pH, thus repelling each other and weakening the protein A-IgG hydrophobic association. That results in elution of IgG from the affinity column. (Shukla, “strategies to address aggregation during protein A chormotography” 36 BioProcess Technical. May 2005).

The basic protocol of protein A affinity column is to bind at neutral pH and elute at acid pH (Fahrner, Biotec. Genetic Eng. Reviews, 18, 2001, p. 309, last ¶). See Affinity chromatography.

Antibody concentration

By using the Beer-Lambert law, the concentration of IgG (mg/ml) in the sample can be measured by multiplying the adsorbance at 280 nm by 0.72. If IgM or IgA are purified, multiply the absorbance at 280 nm by 0.84 or 0.94 respectively. These antibody concentrations are only estimates but can provide a reliable and quantiative method for determining the concentrations of pure antiboyd solutions. Most researchers use a sandwich ELISA assay to accurately measure antibody concentyrations within a range of 1-20 mg/ml sample. Antibodies can also be monitored for purifyt by SDS-PAGE. IgG appears in a reducing SDS-PAGE as 25 kDa and 50-55 kDa bands and IgM appears as 25 kDa and 70-80 kDa bands. Recovery of immunoglobuilins can be quantified by a standard protein assay, scanning densitometry of reducing or non-reducing SDS polyacrylamide gels or ELISA. Proteus “Protein A Antibody Purificaiton Handbook, 2005).

Hober (J. Chromatogr B 848 (2007) 40-47) discloses that the capacity amoung several commercial SPA medi resins ranged from 0.5 to 20 mg/ml and that for large scale applications, optimisation incdes dynamic capacity, antibody concentration in the load, the number of column volumes for elution/equilibration/wash, the load flor rate and the elution/equilibration/wash flow rate.

–Polycarboxylic acid

Ngo (US4,933,435) teaches purifciation of immunoglobulins on Protein A adsorbent at pH of 6-10 and containing at least one polycarboxylic acid which is any organic acid having more than one carboxyl group, including salft forms thereof. Among the numerous carboxylic acids listed are acetate, glycine, aspartate, gluatamine, malate, glutarate, succinate, Tertrate, EDTA.

–Salts

      –—-NaCL: Millipore “Affinity Chromatography Media” 2004, teaches that optimal binding for monoclonal antibodies is with 50 mM phosphate and 0.15M NaCl pH 7.5.

It is assumed that binding between IgG1 and protein A is at least partly based on hydrophobic interactions so that conditions which favour these interaciton should also enhance the binding of IgG1 to protein A. Accordingly it has been disclosed that increasing the pH and/or the ionic strenght of the binding buffer enhances the affinity of these mabs for protein A. (Van Sommeren (“Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to prtoein A sepharose 4 fast flow” Preparative Biochemistry, 22(2), 135-149 (1992), showing high dynamic binding capacities even at neutral pH with 0.1 M tris 1.5M(NH4)2SO4 and 0.1 M tris 1.0M Na2SO4 buffers (pH7.5). 

      —–Kosmotropes (Katropic Salts):

The term “kosmotrope”(order-maker)  denotes solutes that stabilize proteins. An alternative name for kosmotrope is “compensatory solute” as they ahve been found to compensate for the deleterious effects of high salt contents (which destroy the natural hydrogen bonded network of water) in osmaotically stressed cells. They should be distinguished from “chaotropes” (disorder-maker) which denote solutes that destablize proteins. (Jungbauer (US8,058,410).

Ngo (J. of Immunoassay & Immunochemistry, 29(1), 2008, 105-115) disclose that kosmotropes (kosmotropic salts) such as ammonium phosphate, ammonium sulfate and sodium sulfate, enhance the yield of antibody purfied by Protein A chromatography. Ngo disclsoes purification of immunoglobulin on protein A using a buffer having a pH 7.5-10 and containing a combination of monovalent cations such potassium ions and polybasic anions such as phosphate ions. These monovalent cations and polybasic anions  ions can be provided by the use of potassium phosphate or ammonium phosphates and ammonium sulfates and sodium sulfates.

Wang (13/898929, 13/898984 and 14/085503) teaches using a kosmotropic salt such as ammonium sulfate, sodium sulfate, sodium citrate, potassium sulfate, potassium phosphate, sodium phosphate, to improve antibody binding onto a Protein A resin. The kosmotropic slat contributes to teh stability and structure of water-water interactions and causes water molecules to favorably interact with proteins and stabilize the intermolecular interaction, thereby increasing the retention of the antibody of interst on the protein A resin. 

–Effect of Temperature: Yityoong (J Immunological Methods, 109 (1988) 43-47) disclose that certain mouse IgG subclasses that bound weakly to protein A at ambient temperature would bind more firmly if the temperature of the system was lowered to 4C. At this temperature, irrellevant proteins could be washed off easily.

When adsoprtion buffers of relatively low ionic strenght are used, improvement of the binding of IgG1 antibodies to protein A can also be obtained by lowering the temperature (Van Sommeren (“Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to prtoein A sepharose 4 fast flow” Preparative Biochemistry, 22(2), 135-149 (1992). 

Wash

Non-specific binding of mAbs to protein A affinity media is usually due to either ionic or hydrophobic interaction with the base matrix or immobilization chemistry. Glass based matrices, for instance ProSep®-A, may exhibit relatively higher HCP levels compared to agarose based matrices. This is htought to be due to higher non-specific binding inherent to the glass matrix ompared to agarose (IP.com Number : IPCOM000127319D, August 22, 2005). 

The aim is to modify the post load wash buffer in such a way as to disrupt these interactions, which will elute the non-specifically bound contaminants without prematurely eluting the mAb. Several approaches have proven effective which include selecting a pH for the intermediate wash buffer that is between the loading and the elution buffers, and/or inclusion of salt, detergents or amino acids such as arginine (Millipore Technical Brief “Increasing Purity of ProSep®-vA affinity chromatography media using an intermediate wash step” 2006. 

Millipore “Affinity Chromatography Media” 2004, teaches that wash buffers usually have the same buffer components as the loading buffer (see above) to ensure the captured product remains bound to the column while non-binindg contaminatns are washed off. However, in the case of non-specific binding due to ionic interactions, adding salt (1M NaCl) has been found to be effective. 

Multiple washing

–-Using differing pH and/or conductivity:

Mendiratta (WO 2014/207763) discloses a method for the purification of mAb from cell culture which includes Protein A with several wash buffers. The second wash is conductited at a pH and/or conductivity higher than the first wash and the third wash at a pH and/or conductivity lower than the second wash. 

Buffer additives: (amino acids):

—Arginine:

Barron ( http://www.priorartdatabase.com/IPCOM/000127319 ) describes an intermediate wash solution for Protein A chromatography containing 0.5 to 2.0 M arginine in a phosphate/acetate buffer at pH 5-7.5 which is used to remove HCP contaminants. They also tested an intermediate wash which contained sodium chloride at 0.5-2 M at pH 5.0-7.5 but reported that NaCL wash showed no significant decrease in HCP.

PCOM000127319D, August 22, 2005 also discloses that introducing an intermediate arginine wash step before elution of ProSep-vA high capacity column can significanlty decrease the aount of HCP non-specific binding caused by a model CHO feedstock. 

Eckermann (US13/522030, now US 9,284,347) disclose a method of depleting impurities from a solution that contains a protein which comprises the Fc domain of an immunoglobuilin by protein A chromatography using a wash buffer fwhich contains 1) arginine, 2) sodium chloride 3) an alcohol such as isopropanol, n-propanol and ethanol and 4) polyvinylpyrrolidone and/or a detergent.

Ritzen “J. Chromatography B, 856 (2007) 343-347 reports that washing of a mAb bound to an affinity resin with 0.5M arginine is an effective way for specific removal of protein bound endotoxin. 

Sun (US20080064860 and 20080064861 see also WO2008/031020) also describe washing that conntains arginine at a concentration of 0.1-2.0 M and pH 4.5-8.0. Bing (US2011/015730) also discloses an intermediate washing of an affinity matrix where the wash comprises and equilibrating buffer having similar pH and sale concentrations as the feed and further compirsing an elevated concentration of salts (e.g., sodium chloride). In some embodiments the wash may contain one or more charged amino acids such as arginine. 

—-Arginine + High pH

Frauenschuh (US 13/516960, now US 9505803; see also 15/336878, published as US 20170044211; see also US 16/363,170, published as US 2019/0233469) disclose a wash solution for affinity chromatography which uses arginine and a nonbuffering salt such as a halogen salt such as sodium chloride (NaCL), magensium chloride (MgCl2) and potassium chloride (KCl), preferably at high pH above 8.0. The combination of arginine and a nonbuffering salt is reported to remove more impurities than wash solutions containing either arginine or salt alone and results in a sharper elution peak correlating with a high concentration of the protein of interest. The term “nonbuffering salt” does not include buffering salts such as sodium acetate, sodium phosphate and Tris, which contribute to retaining the pH of a wash solution udner the applied conditions. However, Frauenschuh (US 14/122,707, published as US 9,663,552) discloses a wash buffer peromred without the presence of a nonbuffer salts where the wash buffer includes arginine at a pH at least 8.5. 

—-Arginine + guanidine:

Holstein (“Protein A Intermediate wash strategies” BioProcess International, 13920, 2015) disclsoes that increasing arginine in a Protein A intermediate wash buffer dramatically reduced the level of HCP. Increasing GuHCL concentration also eld to less HCP in the elution pool. In addition, an intermediate wash consisting of 0.05M Tris, 2 M GuHCL, pH8.5 led to the loest HCP in the elution pool. 

Wang (US 15/659093, published as US 2018/0021696) disclsoes  method for preparaing a protein of interest using a wash buffer for Protein A that includes a basic amino acid such as arginine and guanidine. 

–Caprylate:

Gruber (WO0214186350) discloses a method for separating a recombinantly produced polypeptide such as an antibody from HCP using a Protein Chromatography column which includes a washing buffer that has between about 50-100 mM sodium caprylate at pH 8-9. In one embodiment, the wash buffer includes about 100 mM sodium capyrlate in 100 mM Tris at a pH of about 9 and about 2.5M sodium chloride. 

—-Arginine + caprylate

Aboulaich “A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies” Biotechnol. Prog. 2014, 30(5)) discloses identifying HCPs that assocaite with mAbs by imobilizing mAb onto chromatography resinfollowed by incubation with HCPs obtained from supernatant of non-mAb producer cells The dissociation of the HCPs form the mAb using various washes were identified using mass spectometry. A combination of sodium capryalte and arginine as a wah buffer was more beneficial than with arginine alone and reduced the levels of a alrge number of HCPs to undetectable levesl (0%).  Aboulaich disclsoes that this approach could potentially facilitate rapid development of wash condtions to improve HCP clearance during the Protein A capture step for mAb mnufacturing. 

Dumetz (US 16/330,588, published as US 2021/0284686) discloses a method of purifying an antibody from HCPs such as Phospholipase B-like 2 protien and cathepsin L using a superantigen chromatography solid support such as Protein A with a wash buffer that includes about 50 mM caprylate and more than about 0.5 M arginine. 

–Histidine

Ishihara (EP 2583973, published 8/20/2011) discloses purificaiton of mAbs using Protein A chromatography such as MabSelect SuRe where the column was washed with an equislibration buffer that inlcudes histidine pH 7.0. 

—-Histidine + Tris

Falkenstein (US 15/900,443, published as 2018/0186865) dicloses a wash buffer for use in Protein A chromatography for the purificaiton of IgG4 or IgG1 isotype antibodies that incldues both histidine and tris.  anda.a pH of about 6.5 or higher. In one embodiment, the was step includes a low conductivity aqueous solution. Falkenstein (US 15/900,449, published as 2018/0186866) also discloses a method for purifying a bispecific antibody using Protein A with a wash buffer of low conducitvity. In one embodiment, the buffer aslo includes Tris. In one embodiment, the method further includes an additional wash using a high conductivity which can also include histidine. McDonald (US 15/900, 461, published as 2018/0186832) also discloses a method of purifying a human IgG1 or IgG4 antibody with a Protein A that includes using a wash buffer with low conductivity of 0.5 mS/cm or less. In one embodimetn the low conductivity aqeuous solution has a pH of about 7 or more and includes Tris. 

Adjustment of pH

–Intermediate pH:

Non-specific binding is usually due to eitehr ionic or hydrophobic interaction with the base matrix or ommobilization chemistry. The aim is to modify the post-load wash buffer in wuch a way as to disrupt these interactions, which will elute the non-specifically bound contaminants without prematurely eluting teh MAb. Selecting a pH for teh ntermedaite wash buffer that is between the loading and the elution buffers is one approach to do this. For example, if the loading pH is 7.5 and the lution pH is 4.0, then an intermediate wash buffer pH of 5.0 might be optimal. In teh case where an intermediate pH wash is not practical or is insufficient alone to attain required HCP levels, then the second step is to evaluate the addition of a salt, amino acid, detergent or solvent in the intermedaite wash buffer (see below). Millipore Corporiation (2006) “Increasing purity on Prose. 

—High pH:

Shukla “Host cell protein clearance during Protein A chromatography: development of an improved column wash step” Biotechnol. Prog 2008) discloses a Protein A wash buffer with high pH such as 9.0. A wash buffer consisting of 1 M urea and 10% isopropanol was partciularly effective. 

Wang (US 15/558,033, published as 2018/0078876, published as US 10695693; see also US Patent Application No: 16/881,102, published as US 20200282332) discloses a method of purifying an antibody using an affinity chromatography such as Protein A and a high alkaline pH such as one at least 9.0. 

Addition of Caprylic Acid

–Caprylic Acid

It has been shown that by adding 25 mM carpylic acid to the wash buffer during purificaiton of polyclonal IgG from ovine serum using Protein A mimetics the level of non-specifically bound albumin could be reduced. The purity of IgG could be increased form about 80 to 95% by introducing this intermediate wash after laoding of serum to the column. Monie, “Evaluation of the 96-well format for screening of chromatographic buffer condtions” Master’s Degree thesis, Uppsala, University School of Engineering, October 2006.

—–Caprylic Acid with salt

Gruber (US 14/890,823, published as US 2016/0108084) discloses a method of reducing HCP by loading a cell culture sueprnatant onto a Protein A chromatography column and washing with a buffer that includes 25 -200 mM fatty acid and sodium chloride at a concentraiton of about 1-2.5M. 

—–Caprylic Acid without salt

Goklen (US 14/775868, published as US 2016/0024146) discloses a method for purifying an antibody using Protein A chromatography that includes a first wash buffer that includes 10-125 mM sodium caprylate without the addition of NaCL. 

Jensen (US 2017/0044210) discloses a method for purifying a target protein such as an antibody by loading a sample with the protein onto an affinity chromatography and using 2-10 mM caprylic acid so as to inactivate and way away viruses followed by eluting the target protein. 

—-Caprylic Acid + NaCL:

Gruber (US 14/890823, published as 2016-0108084) discloses that wash buffer at pH 9, with 2.5M sodium chloride and 100 mM sodium cpayrlate improved HCP clearance. 

Hydrophobic electrolyte solvent

Blank (6,127,526) discloses a method for purifying an antibody by Protein A chromatography by adsorbing it onto a solid phase comprising silica or glass such as controlled pore glass column or silicic acid column, removing contaminants by washing with a hydrophobic electrolyte solvent such as TMAC or TEAC and recvoering the antibody.

Salts:

–Salts + detergents, solvents and polymers

Protein A chromaotgraphy wash buffers containing salt (such as sodium chloride) alone or in combination with either a detergent (e.g., tween 20), a solvent (e.g., hexylene glycol) or a polymer (e.g., polyethylene glycol) have been described (Breece, 6,870,034). 

Benzoate Salt + benzyl alchol: Beigie (US 16/228,291, published as US 2019/0233468) discloes that using 0.5 M sodium benzoate pH 7.0 and benzyl alcohol in an intermeiate wash was very effective at removing HCPs such as PLBL2 during Portien A purification. 

tegramethylammonium chloride(TMAC): One way to decrease the amount of host cell proteins in the elution pool when using Prosep media is by using an intermediate wash with tegramethylammonium chloride(TMAC) (Fahrner, Biotech. Genet. Eng. Rev. 18, 2001, p. 311, 2nd ¶).

Urea

–Urea + Isopropanol

Shukla (WO 2007/1009163; see also Biotechnology Progress, 24(5), 2008, pp.1115-1121) disclose that a combination of 1M urea and 10% isopropanol in the wash buffer minimized HCP levels during Protein A chromatography for the purificaiton of antibodies.

–Urea + NaCL

Woo (US 15/924,163, published as US 2018/0265543) discloses a method of purifying an antibody using protein A chromatography wehre a wash solution that includes urea is used. Results showed that a wash solution that includes arginine, guanidine or a comination of urea with sodium chloride, resulted in a reduction of HCP. 

–Urea + Dextran Sulfate

Zhengjian (WO 2016/153978) discloses use of dextran to enhance Protein A chromatography wash. In one emboodiment other wash buffer components such as salt which is sodium chloride and a chaotropic reagent which is urea was combined with dextran sulfate. 

Elution

The most common elution conditions for Protein A or G affinity and immuno-affinity separations involve a reduction in pH to between pH 2.5 and 5.0. “Protein A Antibody Purification Handbook” Pro-Chem Inc. 2005.

Millipore “Affinity Chromatography Media” 2004, teaches teaches that the highest pH which gives acceptable recovery of the target antibody should be used which for IgG is usually pH3.0-4.0. 

A variety of strategies have evolved to address the issue of aggregation/precipitation during Protein A elution such as modification of the elution buffer to make the buffer conditions more conducive to product stability. Stabilizers such as arginine have been added to the elution buffer to reduce aggregation of eluting antibodies. Low temperature operation of the Protein A step can in some cases reduce product aggregation. (Shukla, J. Chromatogr. B 848 (2007) 28-39)

–Addition of amino acids:

——Arginine, histidine and proline:  

Arginine is an effective agent for elution of antibodies from Protein A and G columns (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555). Arakawa (“protein Expression and Purification, 36: 244-248, 2004) disclose eluting antibodies from a Protein A column using an elution buffer containing 0.5-2.0 M arginine at pH 4.1-5.0. Recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 30% recovery (Arakawa, “Elution of antiboides form a Protein-A column by aqueous arginine solutions, Protein Expression and Purificaiton 36 (2004) 244-248). 

(Chmielowski (US13/058301) discloses the amino acids histidine, proline or arginine in elution buffers as stabilization agents for Protein A affinity.

–Acetate: 

Chmielowski (US13/058301) discloses a method for purifying a monoclonal antibody by contacting the sample with Protein A affinity chromatography at a temperature from about 15-27C and eluting with acetate.

Shukla (US 2009/0306351) teaches a method for purifying a monoclonal antibody with an elution buffer of 100 m acetate, pH 3.6. 

–Citrate (citric acid/sodium citrate): 

Chmielowski (US13/058301) discloses a method for purifying a monoclonal antibody by contacting the sample with Protein A affinity chromatography at a temeprature from about 15-27C and eluting with citrate.

Hickman (WO 2010/048192 A2) teaches equilibration with a suitable buffer such as Tris/NaCL, pH around 7.2. Following loading of the column, the column can be washed as for example with 20 mM citric acid/sodium citrate, 0.5 M NaCl at pH of about 6.0 and then eluted with a suitable elution buffer such as  acetic acid/NaCl buffer, pH around 3.5.

Shukla (BioProcess International, May 2005, pp.36-44) teaches various combinations of elution buffers during protein A chromatography of a monoclonal antibody including 50 mM citrate, pH 3.6. Yumioka (EP1563710) teaches a method purifying monoclonal antiboidies on a protein A affinity chromatography which involves eluting in a citrate buffer of pH3.5. The buffer can further comprise arginine.

–pH: Since most antibodies retain the native structure above pH 4.0, various attempts have been made to elute the bound antibodies above this pH (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555).

—-Reducing pH or Temperature or adding Protease inhibitors: 

Fahner disloses a method for reducing leacing of protein A during protein A affinity chromatography by reducing temperature or pH or by adding one or more protease inhibitors to a composition that is subjected to protein A affinity chromatography. In one emboidment the termpature is in the range form about 3C -20C.

Nielsen discloses a method for reducing leaching of protein A by reducing temperature or pH or by adding protease inhibitor(s) to a composition that is subjected to protein A affinity chromatography (WO 2005/016968).

For effects of temperature on protein A chromatography see also Van Sommeren “Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antiobides to protein A Sepharose Fast Flow” Preparative Biochemistry, 22, 135-149, 1992  and Tu “Temperature effects binding of murine monoclonal IgG antibodies to protein A. J. Immunological methods, 109, 43-47, 1988. 

Shukla (BioProcess International, may 2005, pp. 36-44) teaches conducing protein A chromatography at room temperatures (20-25C) but that colder termpatures gave a lower rate constant for aggregation (2-8°C).

—-Neutral or Alkaline pH  or Magnesium chloride

Deepak (IN 183939) discloses prapring pure monospecific polyclonal antibodies to malarial lactate dehydrogenose useful for the diagnosis of mario by adsorbing serum collected from animals having antibodies against LDH on Protein/A separhose column and eluting with a buffer having a pH of 2-5 or 10-11.8 or with a sale such as Magneisu or lithium chlorida or potassium thiocynate (3-5M). 

Tustian (US 2016/0024147) disclose that employing a chaotropic agent and pH gradient or pH step elution buffers results in imporved peak resolution between closely related molecular species. Bi specific antibodies containing aprotein A binding ablating substitution CH3 domain paired with a protein A binding CH3 domain are separated with high peak resultion from monosepcific antibodies containing aprotein A binidng ablating substituted CH3 domain wpaired with a protein A binding ablating substitutedCH3 domain and monospecific antibodies containing a protein A binding CH3 domain paired with a protein A binidng CH3 domain. Useful chatropic agents include magesium chloride and calcium chlroide. In one embodiment the VH binding bsAb was loaded on a Mabsapture A resin and following a series of washes the antibodies were eluted from pH 6 to 3 with magnesium chloride. 

—-Ph Gradients:

Bian (US 14/768254, published as US 2016/0122305) discloses a method of purifying an antibody using a MAbSelect SuRE column using a pH gradient that used a high and low pH buffer. In particular, Bian showed that using two types of modified C domain resins for the Protein A ligan was particularly effective effetive in removing agregates with the pH gradient whereas a large aggregate peak was observed in the fractions eluting towards the end of the profile for MabSelect SurE, a large agreggate peak was observed in botht eh early and late eluting fractions for the modified C domains. 

Brown (13/393525, published as US 2013/0041139) teaches purification of polypeptides having a CH2/CH3 region such as antibodies by binding the polypeptide to Protein A and leuting with a pH gradient starting at a pH of 5.0 using an elution buffer that contains a high and low pH buffer such as acetate and formate.

Davis (US2010/0331527) discloses islation of a bispectific antibody on a solid support comprising Protein A by employing a pH gradient, such as a linear gradient from pH 4.2 to pH 2.8. 

Duhamel, J. Immunological Methods 31: 211-217 (1979) discloses using a pH gradient to elute column bound IgG into at least two major overlapping peaks centered 0.4 pH units apart in the gradient with the first peak centered at pH 4.7 and the seccond at pH 4.3. Upon second passage of each component, the high pH eluting IgG conatined less than 1% IgG1, 95% IgG2 and 5% IgG4 and the low pH eluting IgG contained 90% IgG1, 6% IgG2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. 

Better (US5,576,184) discloses loading of anytibody onto a Protein A colum and elution with a pH gradient (pH2-9). The antibody was found to elute between pH 3.5 and 4.0. 

Ishihara (US 2006/0257972) discloses applying an antibody mixture to a Protein A column and performing a gradient elution where a buffer (pH 2.5) onctianing 0.10 M sodium acetate, 0.10 M glycine and 0.15 M sodium chloride is gradually added to a buffer (pH 8.0), which contains 0.10 M disodium phosphate, 0.10 M sodium acetate, 0.10 M glycine and 0.15 M sodium chloride to gradually lower the pH level (o.e., pH gradient elution). 

Martin discloses that guinea pig serum was chromatography using protein A-Sepharose chromatography in citrate-phosphate buffer at pH 7.3. The bulk of serum proteins eluted in the starting buffer. Two peaks of protein A bound serum proteins were eluted by a decreasing pH gradient, a small peak centered at pH 4.7 and a larger peak centered at pH 4.3. IgG contained in the peak eluted at pH 4.7 has fast gamma immunoelectrophoretic mobility and IgG in the peak eluted at pH 4.3 had slow gamma mobility. 

Pan (US2008/0167450 and WO2008085988) teaches methods of purifying Fc domain containing polypeptides comprising binding said polypeptide to protein A and eluting with a pH gradient such as one where the top of the gradient starts above pH7 and the bottom is at or below 4.0. 

Pan Analytical Biochemistry 388: 273-278 (2009) also disclose that under the typical step gradient mode, protein A tightly binds the Fc portion of teh antibody under neutral pH conditions (pH 7.0-8.0) while impurities are washed away after which the acidic pH buffer (pH 3-4) is used to release the antibody from the protein A column. A pH gradient instead of a typical step gradient mode can also be used. 

–Salt Gradients:

Van Alstine (US 2007/0213513) discloses in a multi step chromatography procedure with affinity chromatography uisng salt gradient elution will require a subsequent deilution step before the next step of ion exchange chromatograph. 

 Cleaning

Millipore “Affinity Chromatography Media” 2004, teaches that the use of a low pH 9pH 1.5) regeneration with phosphoric or hydrochloric acid solution after ever cycle is very effective at removing strongly bound material from ProSep-A media which is protein A immobized on porous glass.