See also Analogues of Protein A (e.g., variants of Protein A which can be attached to the various types of resins below).

Definitions

Cross-Link: is a bond that links one polymer chain to another. They can be covalent or ionic bonds.

column Dimensions/dynamic binding capacity (DBC)/Flow Rate/Pore Size

A Protein A bed height employed at large scale is between 10 and 30 cm, depending on the resin particle properties such as pore size, particle size and compressibility. Flow rate and column dimensions determin antibody residence time on the column. The typical linear velocity employed for Protein A steps ranges from 300-500 cm/hr. Dynamic binding capacity ranges from 15-50 g of antibody per liter of resin and depends on the flow rate, the particular antibody to be purified as well as the Protein A matrix used. (Liu, “Recovery and purificaiton process development for monoclonal antibody productn, mAbs 2:5, 480-499, 2010). 

Biang (EP2014359) discloses suitable solid supports for performing affinity chromatography comprising a silica particle having a pore size greater than 630A and less than 1000A and a mean particle wsize greater than 50um. The support may have a DBC of greater than or equal to 45 g/L at 10% breaktrhough and a pressure drop of less than or equal to 2 bar at a flow rate greater or equal to 400 cm/fr for a packed bed height of 20cm in a column with a diameter of greater than or equal to 3.2 cm.

For rProtein A sepharose Fast Flow manufactured by GE Healthcare, which is a typical first generiton Protein A resin, the ordinry antibody binding capacity is 15-20 g/L resin whereas, for teh second generation resin manufactured by the same company, Mab Select SuRE, which is currenlty most commonly used worldwide, the generally observed bidning capacity is about 30 g/L resin. In addition, compared to the former resink the latter can accommodate a lienar flow rate about 1-5 to 2 times higher and a mroe efficient Protein A purificaiotn of antibody has been possible. Tanaka (Chugai, Tokyo, US Patent application 16/061,454, published as US 2019/0330268)

Specific Types of Protein A Resins

There are three major types of Protein A resins, classified on their resin backbone composition: glass or silica based (e.g., Prosep vA, Prosep Va Ultra (millipore); agarose-based (e.g., Protein A Sepharose Fast Flw, MabSelect (GE Healthcare) and organic polymer based (e.g., polystyrene-divinylbenzene Poros A and MabCapture (Applied Biosystems). The column bed hight employed at large scale is between 10-30 cm, depending on the resin particle properties such as pore size, particle size and compressibility.

Glass or silica based:

–Controlled-pore glass (CPG) beads have also been used. Although high throughputs can be obtained with columns packed with CPG, this carrier is even more expensive than agarose gel beads.

Mechanically stable matrices such as ontrolled pore glass allow for faster flow rates and shorter processing times (Fahrner US2008/0193981).

Commercial examples of Protein A affinity chromatography having Protein A immobilized onto a controlled pore glass backbone include PROSEP A and PROSEP vA media/resin from EMD MILLIPORE. (Bian, US 14/768,254, published as US 2016/0122305). 

Polysaccharide based: such as dextran, starch, cellulose, pullulan or agarose can be used as protein A resins. Such polysaccharides can be produced by known methods (Tamori (US13/636410, referecing Japanese Patent No. 4081143). 

–Agarose Based resins:

In the technique of affinity chromatography, agarose gells and crosslinked agarose gels have been the most widely used support materials. Their hydrophilicity makes them relatively free of nonspecific  binding, but their compressibility makes them less attractive as carriers in large scale processing, such as in manufacturing.

Of the beaded agarose derivatives commercially available, Sepharose 4B (Pharmacia) is the most useful for affinity chromatgoraphy. Chemical compounds containing primary aliphatic or aromatic amines can be coupled directly to agarose beads after activation of the latter with cyanogen bromide at alkaline pH. (Cuatrecasas, “Affinity Chromatography” methods enzymol, 1971; 12: 345-78).

–Cellulose based: 

Cellulose particles have also been used for synthetic affinity sorbents. However, compared to agarose gels, cellulose particles are formed with more difficulty and thus have received less attention in the preparation of affinity sorbents for enzymes. Cellulose, however, is perhaps the least expensive of all support matrices.

Two lesser used support matrices are polyacrylamide gel beads and Sephadex gel beads made from dextran and epichlorohydrin. Although convenient methods have been developed for using them, the softness of these beads yields poor column packings and their low molecular porosity yields a sorbent with poor ligand availability to the ligate. Coupek (US 4,281,233) show supports for affinity chromatography which comprise copolymers of hydroxy aklyk acrylates or methacrylates with cross-linking monomerrs. The copolymers contain covalently attached mono or oligosaccharides.

High capacity protein A media have higher dynamic binding capacities than older versions. MabSelect SuRe was designed to exhibit improved stability in alkaline solutions. Prosep-vA-Ultra exhibits a pore size of 700 A and was also designed to have higher binding capacity due to the smaller pore size and higher surface area compared to Prosep rA. The media MabSelect Xtra and MabSelect SuRe are based on cross-linked agarose and the medium ProSep-vA Ultra is based on controlled pore glass  (Kazumichi, J. Chromatography A, 1102 (2006) 224-231).

Polymer having a hydrophilic surface: includes a polymer having  hydroxyl group (-OH), a carboxyl group (-Cooh), an aminocarbonyl group (-CONH2 or N-substituted type), an amino group (-NH2 or N-substituted type), an epoxy group, an oligo group or a polyethyleneoxy group at the outer surface (and if aavilable, also at the inner surface).

Commercial examples of Protein A affinity chromatography having Protein A immobilized onto an agarose solid support include rPROTEIN A SEPHAROSE FAST FLOW and MABSELECT from GE HEALTHCARE. . (Bian, US 14/768,254, published as US 2016/0122305).

A hgih capacity Protein A resin (MAbSelect SuRE LX) from GE Healthcare with a reported DBC at 10% breakthrough of greater than 60 mg/mL is known.  (Ghose, “Maximizing Binding Capacity for Protein A Chromatography” 2014, American Institute of Chemical Engineers. 

–Pourous cellulose beads with high capacity DBC (5% DBC) not less than 70 g/L

Kawai (US 2015/0297820) discloses porous cellulose beads which further includes an affinity ligand such as protein A with a 5% DBC of IgG for residence time of 3 minutes not less than 60 g/L

Snynthetic polymers: 

The solid support can be based on synethic polymers, such as polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, etc. In case of hydrophobic polymers, such as matrices based on divinyl and monovinyl-substituted benzenes, the surface is often hydrophilised to expose hydrophilic groups to a surrounding aqueous liquid. Such polymers are easily produced according to standard methods (Ander, US14/385336, published as US2015/0080554))

Synthetic polymers include poly(meth)acrylate, poly(meth)acrylamide or a styrene-divinylbenzene copolymer. Such a synthetic polymer can be produced by a known method such as in J. Mater, Chem., 1991, 1(3), 371-374. The amount of Protein A/G immobilized on the support is disclosed as being increased compared to related art supports, such that antibody purificaiton capability can be increased.

Mechanically stable matrices such as (styrenedivinyl)benzene allow for faster flow rates and shorter processing times (Fahrner US2008/0193981).

Commercial examples of Protein A affinity chromatography having Protein A immobilized onto a polystyrene solid phase include POROS 50A and POROS MabCapture A from BIOSYSTEMS, INC. . (Bian, US 14/768,254, published as US 2016/0122305).

–Production Techniques:

Crosslinked polystyrenes are usually produced by suspension (or bead) polymerization in finely spherical form particularly suitable for chemical manimipulation. In troduction of a given functionality inot the polymer beads can be acheived either during, or after, the polymerization. In the former, appropriate mixture of styrene, divinylbenzene and a suitably chosen functional or reactive styrene derivative is copolymerized to produce the functional group carrying resin direclty form the polymerizaton process. The alternative strategy involved the copolymerization followed by post-polymerization functionalization of the crosslinked polymer. (Arshady, “Styrene based polymer supports developed by suspension polymerization” Chimica e L’Industria, 1988, 70(9): 70-75). 

Cross-linking of Resins and Additional Constituents

In order to realize higher flwo rates, chromatography packing materials produced using a technique of crosslinking a base gell to icnrease its strengh have been developed. The corsslinking agent is not particularly limited and includes epichlorohydrin, polyethylee glycol or an epoxy compound made by glycidyl-etherifying hydroxyl groups of sorbitol,  since bond with cellulose is chemically stable and charged groups which may cause undesired adsorption action are not introduced. These crosslinking agents can be used solely or in combination. Matsumoto (US 2011/0301330)

 Cross-linked gels with hydrophilic polymers:

 It is known that adsorption characteristics are improved by combining these corss linked gells with a hydrophilic polymer such as dextran. Matsumoto (US 2011/0301330)

Matsumoto (US 2011/0301330) discloses a porous cellulose gel for antibody purificaiton which is made by adding polysaccharides such as dextran and pullulan haivng a limiting viscosity of 0.21 to 0.90 dL/g to porous cellulose particles, the dry weight per uit volume of the porous cellulose gel being 1.06 to 1.40 times the dry weight per unit volume of the porous cellulose partciles. When the surface or the inside of porous of the porous cellulose partciels in which cellulose molecules are crosslinked by a cross linking agent such as epichlorohydrin is modified by polysaccharides having a predetermined limiting viscosity, high flow rate characteristics are ensured in use for a chromatography packing material, and a ligand can be efficiently introduced, and therefore, high adsorption characteristics can be provided. 

Methods of Coupling Protein A Ligands to Solid Resin:  See Outline

Commercial Types of Resins

See Hober (J. Chromaogr. B 848 (2007) 40-47) Table 2 for list of protein A based affinity media and their manufacturer. 

Recombinant Protein A and native protein A have the domain structure EDABCX, MabSelect has the domain structure EDABC and Mabselect suRe has the domain structure ZZZZ. (Ariane Marolewski, “Quantiation of MabSelect Sure Protein A ligand: why a matched standard matters, Repligen)

Regular protein A media based on native or recombinant protein A (.e.g, MABSELECT and rProtein A SEPHAROSE 4 Fast flow) normally elute at pH 3.1-4.0 mianly depending on its VH3 binding. The alkaline stabillized product MABSELECT SURE, erived from the B domain of portein A, essentially lacks the VH3 binding giving a higher elution pH: 3.7-4. (Ander, US14/385336)

Examples of Commercial Resins:

 Resin Vendor                Ligand/Matrix        Vendor Matrix           References                                                 

MabSelect

MabSelect Protein A is an affinity resin composed fo a highly cross-linked agrose matrix that is covalently derivatized through a thioether linkage with recombinat Protein A produced form Escherichia coli (E. coli). MabSelect is a commercially vailable resin contianing recombinant SpA as its immobilized ligand. The specificity of Protein A ligand to the binding region of IgG is similar to that of native Protein A. MabSelect SuRe columns have a similar highly cross-linked agarose matrice used for MabSelect but the ligand used is a tetramer of four idnetically modified Z-domains. Brown (US Patent Applicaiton 16/142,198, published as US 2019/0144531)

MABSelect has a traditional SpA ligand comprised of all five domains E, D, A, B, C while SuRE has a genetically modified Protein A ligand comprising only B domains. (Ghose “Antibody variable region interactions with protein A; implicaitons for the devleopment of generic purificaiotn processes” 2005).

Brown discloses using MabSelect for the purificaiton of ATN-103, which is.a travelent nanobody molecule targeting TNFalpha and HSA. 

MabSelect SuRe          Alkali-stablized protein A derived ligand/agarose

MabSelect SuRE 1. provides greater atbility under alkaline conditions used in clieaning in place protocls 2. Stands for superior resistance; improved stability in alkaline solutions. Exhibited the highest stability compared to MabSelect Xtra (also GE Health-care) and ProSep-vA Ultra (Millipore).

The ligand fo rMabSelect Sure is based on a genetically engineered variant of the Z domain where a number of Aspargines have been replaced with other amino acids. It is a tetramer construct with four identical domains. The ligand is single point attached to MabSelect base matrix. (Simon Jones “Evaluation of an alkali stable protein A matrix versus protein A sepharose fast flow and considerations on process scale-up to 20,000L” Lonza, presented October 2004. 

In Mab Select Sure, domain B of protein A has been modified by genetic engineering to have a tetrameric structure. Mab Select Sure lacks affinity for the antibody variable region and is advantageous in that it allows for antibody elution even under milder conditions as compared to conventional recombinant protein A. In addition, the resin has improved alkline resistance and enables for cleaning in place using 0.1-0.5 NaOH.  (Igawa (US 2021/0292360)

SuRe was developed to withstand stronger alkaline conditions. Using protein engineering techniques, a number of asparagine (the most alkali sensitive amino acid) residues were replaced in the Z-domain of Protein A and a new ligand was created as a tetramer of four identical modified Z-domains (Healthcare GE, 2005; Ghose “Antibody variable region interactions with protein A; implicaitons for the devleopment of generic purificaiotn processes” 2005). 

–binding to VH region:

Z-domain based protein A variants (like Mab Selecdt Sure) lack the ability to bind to human VH3 domains and as such can not be used for Fab purificaiton (Hermans, US 2013/0337478)

However, Ghose “Antibody variable region interactions with protein A; implicaitons for the devleopment of generic purificaiotn processes” 2005) discloses that differences in elution pHs among the different molecules are significanlty reduced on the SuRe resin which indicates that variable region interactions do indeed play a significant role in determining the elution pH for antibodies on Protein A materials. 

MabSelect Xtra

1. designed to have higher binding capacity than MabSelect.     1. Hahn J. Chromatography A, 1102 (2006) 224-231

rProtein A FF            Agarose         GE Healthcare

 Protein A Sepharose     native protein A                 Amersham Biosciences/ GE Healthcare (e.g., Protein A SEPHAROSE)

 rProtein A Sepharose     Amersham Biosciences/ GE Healhcare (e.g., rProtein A SEPHAROSE)

StreamlinerProteinA   1. .thioester single point attached recombinant protein A  1. US 6,399,750

Prosep A     Protein A ligand attached to CPG matrix by multiple covalnet bonds thoughy lysine side chains on protein A/ porous glass 

Prosep-vA-Ultra  pore size of 700 A      higher capacity due to the smaller pore size and surface area  Millipore

Elution by Change from Neutral to Acidic pH

Elution of the target molecule form the Protein A column generaly requires drastic conditions, a step gradient to 0.1M Glycine-HCL (pH<3.1) is typical. Elution at low pH contributes to virus inactivation, but threatens the yield of biologically active antibody (Schubert, J. Chromatography A, 1142 (2007) 106-113). 

(i) pH/Acids:  It has been shown that a conserved histidyl residue in the center of Protein A binding of IgGs faces a complementary histidyl residue on Protein A. These residues take on a positive charge at low pH, thus repelling each other and weakening the Protein A-IgG hydrophobic association. As a result, operating conditions for Protein A columns typically require the use of low pH conditions (typically between pH 3 and 4) for column elution (Shukla, J. Chromatography A, 1171 (2007) 22-28). 

For elution at pH <3, either citrate or acetate may be used.   The protein A column may typically be run in 4-10 cycles to purify a single batch. Since each cycle is only about 1 hour long, this cycling allows rapid throughput while reducing the cost of the column ((Fahrner, Biotechnol Genet Eng Rev, 2001, 18, 301-27 “Industrial purificaiton of pharmaceutical antibodies: development, operation, and validation of chromatography processes).

Cunningham (US 6800740) teaches purificaiton of MAbs from mouse ascites fluid by bidning to Protein A Sepahorse and elution with 0.1 M acetate (pH 3.0).

Fahrner (US 7485704 and US 2005/0038231) disloses a method of purifying a protein such as an antibody by protein A affinity chromatography and eluting with 0.1 M acetic acid.

Zhou (US2008/0132688) discloses passing a culture media containing Mab over protein A column and elution using a low pH buffer (for example, pH 3.4, 50-100 mm acetic acid).

(ii) polyols: Following the recovery of antibody bound to protein A with an acidic elution buffer solution, a stabilizing agent in the fomr of a polyol compound such as polyethlene glycol, polyvinyl pyrrolidone or ethylene glycol to suppress aggregation has been added (EP 11568710).

Elution by Adding Additives

(i)amino acid addition: 

–Arginine: Yumioka (EP 1568710; see also US 2005/0176109 and US 2012/0142901) teaches a method of antibody purification with Protein A affinity by contacting the antibody with a buffer solution adjusted to pH 4.0 to 5.0 comprising arginine and/or a derivative used to desorb/elute the antibody from the column. Ejima (Analytical Biochemistry 34, 2005, 250-257) also disclose protein A affinity chromatography is enchanced significantly by using arginine as an eluent. Arakawa (Protein Expression and Purification 36 (2004) 244-248 also discloses that the recovery of antiboides was greatly increased with 0.5M arginine and more so with 2M arginie compared to the conventional eluent citrate.

(ii) Mutants of Protein A:  See outline (e.g., temperature sensitive Protein A mutatns). 

Operating Conditions for the Binding/washing/elution

Binding:

(i) pH: It is known that binding of mouse IgG to protein A-Sepharose is pH sensitive and that optimum adsorption occurs using 0.1M sodium phosphate, pH 8.0 buffer (Ngo, 4801687). It has been known for some time that the binding of murine IgG1 to protein A is dependent on the pH. In contrast to the other murine IgG subclasses, complete binding cannot be achieved if the pH is less than 8.0. Recommendations have been made that binding is performed at pH values considerably higher than 8.0. Schuler (J. Chromatogr. 587(1), 61-70 (1991), however, did not find that raising the pH value from 8.2 to 8.7 gave any imporvement in yield at the saline concentrations and temperatures used in that stufy.

(ii) sodium chloride + glycine/ phosphate or Tris: Buffers with a very high concentration of saline are often recommended to strengthen the binding between protein and murine IgG, in particular, IgG1. Examples tof these concentrations are3M sodium chloride plus 1.5 M glycine, 2 M NaCL + 2 M glycine, 0.5 M phosphate or 1 M Tris. Improved binding is achieved by the intensiifcation of hydrophobic interaction (Schuler (J. Chromatogr. 587(1), 61-70 (1991).

Ngo (4801687) teaches a process for the purification of antibodies which includes the step of mixing the meidum containing antibodies with a buffer solution having a pH in the range of about ph7.5 to pH 10 which contains a hydrophobic interaction promoting salt in a concentration of about 0.6M to 1.75M and then containing the resulting buffered medium with protein A to adsorb. Any buffer may be used to provide the desired pH. For example, glycine buffer, borate buffer or trsi buffer can be used in the range of about 0.01M to 0.25M.

(iii) Temperature: has the most significant effect on the strenght of binding which may occur by weakening hydrophobic interactions. With a weakening of the intramolcular hydrophobic bonds, slight changes may be induced in conformation in either one or both binding partners which resutls in an overall increase in affinity. It is thus possible to effectively purify murine IgG1 mabs from culture supernatants using protein A affinity chromatography with a colled column Schuler (J. Chromatogr. 587(1), 61-70 (1991).

Wash: 

–Aliphatic carboxylate (e.g., sodium caprylate, sodium decanoate or sodium dodecanoate):

Goklen (US 14/775,868US2016/0024146; see also US Patent Application No: 15/931,062, published as US 20200277330) discloses a method for purifying a protein using Protein A chromatogrpahy by absorbing the protein to the Protein A and removing a contaminant using a wash buffer that includes an aliphatic carboxylate such as sodium caprylate, sodium decanoate or sodium dodecanoate. In one embodiment the buffer further includes sodium acetate. 

–Arginine:

Sun (US 20080064860 and 20080064861; see also WO/2008/030120) disclose washing affinity chromatography colun with a wash buffer that contains arginine). 

Frauenschuh (US 13/516960 and US2012/0283416, now US 9,505,803) disclose a wash solution for affinity chromatography using both arginine and a nonbuffering salt, such as a halogen salt (e.g., sodium, potassium, calcium or magnesium chloride). . Preferably the wash solution is at high pH. 

Eckermann (US13/522030, published as US 9,284,347) disclose a wash buffer for protein A chromatography which contains 1) arginine, 2) sodium chloride 3) an alcohol such as isopropanol, n-propanol and ethanol and 4) polyvinylpyrrolidone and/or a detergent.

Morii (EP0333474) discloses removal of endotoxin contaminants from an aqueous solution of a valuable protein such as an antibody using affinity chromatography (does not disclosve Protein A in particular) which includes a wash with pH in the range of 4-11 and an amino compound such as arginine at a concentration 1-500 mM.

–Chaotropic agents/ detergents (e.g., Urea) : are protein denaturants that dissociate hydrogen bonds and affect the termtiary sturcture of the proteins. Shukla (WO 2007/109163)

Shukla (WO 2007/109163) discloses a method for purifying a protein using Protein A chromatography taht includes a wash buffer that includes haotropic agents, hydrophobic modifiers and agents that reduce electrostatic interactions for removing host cell contaminants. Examples include urea, guanidinium hydrochloride and sodium thiosulfate. Getergents are examples of hydrophobic modifies that act by competing for hydrophobic sites and inlcude noionic surfactants such as Polysorbate 20 (Tween 20) and Polysorbate 80 (Tween 8), Triton, SDS. sodium larel sufate. 

–Salts: 

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 (UBreece, 6,870,034). 

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 ¶). 

–No Salts: (only Tris base, acetic acid & sodium acetate)

Goklen (WO 2012/135415) discloses equilibrating a Protein A with a buffer that includes 55 mM tris base, 45 mM acetic acid at about pH 7.5, (b) adsorbing the protein from the contaminated solution to the Protein A and (c) removing contaminants by washing with a wash buffer that includes 55 mM Tris Base, 45 mM acetic acid, 300 mM sodium acetate at about pH 7.5 and recovering the protein with an elution buffer that includes 1.8 mM sodium acetate, 28.2 mM aetic acid, at about pH 3.6 wherein all the buffers are made without the addition of NaCl. 

Elution:  See outline

Operating Conditions for the Entire Procedure

 Ligand Density:

Berg (WO2006/065208) discloses a matrix to which antibody binding protein ligands such as Protein A, G and/or L have been immobilised. In a preferred embodiment, the ligands comprise a monomer, dimer or multimer of Protein A domains (e.g., one or more of Domain A, B, C, D and E domains or a dimer or multimer of Protein Z). The ligands may be immobilised using well known methods such as epoxi coupling. In a specific emobdiment, the ligand density is in the range of 5-10 mg/ml. 

 Temperature/Protease inhibitors: 

Fahrner (US 7485704 and US 2005/0038231) disloses a method for reducing leaching 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 embodiment the termpature is in the range form about 3C -20C.

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

Commercially available Temperature Response Protein A resins:  Nomadic Byzen Pro

In General

Koguma (US13/996385; see also US application No. 14/408,458) discloses antibody purification using a temperature responsive protein A medium such that low pH treatment is avoided in the elution or any virus invactivation. This is disclosed as advantageous because damage to the antibody can be avoided and the antibody will have better binding affinity that conducting the protein A affinity using low pH treatment. (Koguma (j of chromatoraphy A1305 (2013) 149-153 disclsoes taht a mutant protein A (thermo-repsoive protein A) bound IgG at 5C and rlease it at 40C and that IgG was purified mainly as a monomer; the TRPA column showed almost a single monomer peak and a small shoulder peak. On the other hand, IgG purifed by a coventional column had several molecules species, possibly corresponding to aggregated IgG1. 

Sato (US2010/0168395 and WO2008/143199) discloses mutatns of Protein A based on the B domain of Protein A whose binding properties change based on temperature which enables immunoglobulines to be bound and washed in a low temperature range and then released in a higher tmerpature range due to the change in the structure on the Protein A. This is advantageous because it enables the antibody to be extracted while the pH is kept at neutral. In other words, one does not need to use a low pH to elute the antibody, a change in temperature can accomplish this goal. 

Conditions:

Elution additives:

Kawooya (US 16/093,994, published as US 2019/0127418) disclsoes a method of purifying an IgG-svFv binding protein using a temrpature senstive Protein A resin where the elution buffer contained /75M NaCL, 0.5 M argine, 05 M proline, 2.2 M sorbitol and 4M urea at pH 7.2

Linkers/Spacers/Graft Chains for Temperature Sensitive Mutants

Kazuo (US 14/410616) discloses a temeprature sensitive mutant based on the B domain derived from protein A which includes a linker sequence having an amino acid sequence that does not comprise a Val-Pro-Arg sequence and is composed of 7-12 amino acid residues. In one embodiment the linker is Ser-Ser-Gly-(Xaa)-Met wherein n represent and interger 3-8 and an n number of Xaa each independently represents a glycine, serine, histidine, leucine or arginine. In another emobdiment the linker is Ser-Ser-Gly-Leu-(Xbb)m-His-Met wherein m represents an interger 1-6 and and an m number of Xbb each idenpendently represents a glycine, serine or arginine residue. 

See also how to attach Multi-domain ligands to the resin

Berg (WO2006/065208) discloses a matrix to which antibody binding protein ligands such as Protein A, G and/or L have been immobilised. In a preferred embodiment, the ligands comprise a monomer, dimer or multimer of Protein A domains (e.g., one or more of Domain A, B, C, D and E domains or a dimer or multimer of Protein Z). The ligands may be immobilised using well known methods such as epoxi coupling. In a specific emobdiment, the ligand density is in the range of 5-10 mg/ml. 

Berg (US 2006/0134805) also discloses monomer, dimer or multimer of Protein A domains such as the A, B, C, D and E, preferably B or C domains. In one embodiment, such a dimer or multimer comprises Protein Z, which is the mutated form of Domain B.

Godavarti (WO 2006/138553 A2) teaches methods of purifying polypeptides having an Fc region such as antibodies or antibody fusions by adsorbing the polypeptides to Protein A or Protein G, followed by a wash with a divalent cation salt buffer to remove impurities and subsequent recovery (pH 2-4). In various emobdiments, the Fc binding protein comprises one or more Protein A and Protein G. 

Gore (WO92/09633) discloses a polypeptide which has at least 2 but not more than 4 binding domain which possess a high degree of sequence homology with the binding domains of SpA , each capable of binding to the Fc region of IgG.

Jendeberg (J. Molecular Recognition, 8, 270-278 (1995) disclsoes by increasing the valency of an analogue of SpA, going from one to two domains, the affinity for Fc1 increases by almost two orders of magnitudesbut no significant increases in affinity was detected going to five domains. Jendeberg describes four Z mutant proteins (L17D), (N28A), (I31A) and (K35A) which decreased affinity to human IgG1 and CD4-Fc1.

Kihira (US 5,580,788 and EP 0550771A1) examined in detail the relationship between the IgG binding ability of the repetitive protein subunits of protein A (E, D, A, B and/or C) such as repetitive units of the AB domain and found that the binding capacity of the repetive proteins for IgG increased in proportion with the increase in the number of repeat of the IgG binding domain (p. 5, lines 51-56; claims 1-4). Kihira also diclsoes that 1-6 number of such repeats (Table 1) and that the methods used may be used in the case of the other domains to easily produce the desired repetitive proteins between identical or different domains, and thus obtained repetitive proteins may be used to purify IgG to a high degree (Example 3, p. 6, lines 30-34). 

Lee (WO95/06125) discloses a peptide comprising binding domains of protein A and protein G (at least one binding domain from each protein). In one embodiment, a gene cassette which encodes for binding domains of both Protein A and Protein G is provided. 

Ljungberg (Mol Immunol. 1993, 30(14): 1279-85) discloses intact as well as SpA derived fragments cotnaining 1-5 IgG binding domains of different compositions and that while all the proteins bound to IgG, regardless of size or composition, the binding strengh differed significantly. Proteins containing 5 domains have a stronger affinity for IgG than those containing 1-or 2. 

Particular Domain Combinations

A-B Domains:

Kihira (US 5,580,788 and EP08632100 discloses linking the A and B domains of SpA which results in superior ability to purify IgG.

C Domain:

Bian (US 16/113403, pulbished as US 2018/0362595) disclsoes two or more C domains of SpA attached to a chromatography resin at more than one site on the resin where each C domain includes the WT C domain having a mutation to replace the glycine at postion 29 with an amino acid other than alanine or tryptophan that reduces Fab binding. 

Hall (WO2008/039141) discloses at least two Domain C from SpA as a multimeric chromatography ligand.

Majima (US 14/916,316, published as US 2016/0215027) also teaches a multimeric immunoglobulin binding protein having 6-10 total domains from SpA such as the C domain having the general structure (R1)n-(R2m) or (R2m-(R1)n where the R1 domain is an amino acid sequence in which a non-lysine amino acid has been replaced such as the lysine being substituted with a non-lysine amino acid at 1-3 of positions 4, 7, and 35 R2 is an immunoglbulin binding domain coccuring at the N-temrinus or the C-terminus of the protein and includes an amino acid residue that covalently bonds to an insoluble support and the R2 domain is based on an amino acid sequence in which the lysine residue(s) are substituted with a non-lysine amino acid only at position 35, or at posiiton 35 and one or more positions 4, 7 and 35. In one embbodiment the R2 domain includes substitutions of 1-6 amino acid reisdues with lysine at positions 40, 43, 46, 53, 54, and 56. Because only the (R2) domains are selectively immobilized on the support via a covalent bond, it is possible to acheive a highly selective immobilization reaction trhough for example a lysine or a cysteine residue. The R1 in trun has an amino acid sequence that does not contain an amino acid that is active to the chemical reaction used for immobilization. When the immobilization reaction used to immobilzie the prtoien on the support takes place via an amino group, an R2 domain can be produce by substituting the lysine residues contained in the amino acid sequence with non-lysine aino acids only in lysine residues occurring at positiosn that interfere with bidnign to an immunoglobulin upon immobilizing the protein on the support and by substituting some of the non-lysine amino acids not involved in bidning to an immunoglobulin with lysine. In the case of immobilization via a thiol group, a new cysteine residue can be added to the R2 domain. A support immobilization reaction using a disulfide bond or a maleimide group that is highly selective to the thiol group may be used for said immobilization.

–Commercially available: 

Toyopearl AF-rProtein A-650F: is a tetramer of the non-Fab binding C domain which exhibits DBC of greater than 30 g/L for human IgG at 2 minutes residence time. The resin is avialble in 45 um F grade particle size. It is availabe from TOSOH. 

Z domain:

–Z4: MabSelect SuRe (GE Healthcare) is a commercially available Protein A stationary phase that incorporates a ligand, designated Z4, comprising four repeats of the Z domain engineered to be alkaline resistant. A linker with a C terminal cysteine is added to the last repeat to facilitate coupling to the resin. (Pabst, J. of Chromatography A volume 1362, 2014)

Multimers with Asparagine Substitutions in the Domains

Hober (US2006/0194955 and WO 03/080655) teaches an immunoglobulin binding protein such as Staphylococcal protein A (SpA) derivative/analogue wherein at least one asparagine residue of the B-domain and protein Z has been mutated to amino acids other than glutamine or aspartic acid (i.e., such as lysine and leucine) resulting in a ligand with higher binding capacity under alkaline conditions (see above). Hober also teaches that such protein monomers can be combined into multimeric proteins, such as dimers, trimers, tetramers, pentamers etc.

Multimers with Lysine Deletions/Substitutions in the Domains

Majima (EP1992692A1 and US 12/280221, published as US 2010/0286373; also disclose that in the case of using an immunoglobulin-binding protein derived from protein A as an affinity chromatography ligand for an immunoglobulin, a multimer protein abtained by ligating two or more, desirably about 4 binding domains have been conventionaly produced and used. Majama further disclose a modified C-domain of SpA or Z-domain which has improved orientation for maintaining affinity for an immunoglobulin and improved chemical stability under acidic pH conditions because a ratio of the number of lysine at positions 39 onwards to the number of lysine at position. Majima (US 14/916,316, published as US 2016/0215027) also discloses six or ore domains of the C domain with lysine substitutions. 

–In the ligation sequence

Yoda (US 15/669498, published as US 2107/0333811) discloses one of any of the domains of Protein A (E, D, A, B, C and Z) wherein in the amino acid sequence of at least one domain one or more lysines are included and the C terminal lysine is deleted or substituted. In a particular emobdiment, one or more lysines are includes and lysine at position 4 and the C-terminal lysine is deleted or substituted with a hydrophilic amino acid. Specifically, then the C terminal of each domain and a sequence at positions 1-5 in the amino acid sequence of each domain are defined as a ligation site sequence, the C terminall lysine and lysine at position 4 in at least one of the ligation site sequences are dleted or substituted. The amino acid at position 4 is the fourth amino acid from the N teminus of each of the B, C and Z domains of Protein A is lysine. The E domain is treated by assuming that the amino acid residue at postiions 1 and 2 are deleted based on the region with a high homology with the other domains. The D domain is treated by assuming that the third to fifth amino acid reisdues from the N teminus are inserted based on the region with a high homoloy with the other domains. In other wordds, the lysine at position 4 of the domain closest to the N temrinus or the C terminal lysine of the daomin closest to the C terminus is deleted or substituted. By doing this, it was found that by forming a mutated ligation site sequence such that specific lysine (K) which is present in the ligation site sequence is not included, the cleavage of the protein by a serine protease can be avoided. Lysine (K) and arginine (R) present other than in the ligation site sequence, however, are less likely to be cleaved by a serine protease and thus may be present in each domain. 

Ligation of Domains (linkers/spacers):

Linkage of SpA domains:

Honda (US 9,382,297) discloses a modified protein of an extracellular domain of protein A which has the reduced ability to bind to immunoglobulin in an acidic region. Teh amino acid sequence of the modified protein may be a tandem type amino acid sequence in which the amino acid sequence and aribtrary linker sequences are alternately arranged as a plurality of repeats. Such a sequence might be for example (amino acid sequence a -linker sequence A – amino acid sequence a-Linker sequence B -amino aequence a or it could be amino acid sequence a- linker sequence C amino acid sequence b -linker sequence D – amino acid sequence c. 

Rodrigo (WO 2017/1494596) discloses an Fc binding polypeptide with improved alkli stability which include a mutant of a Fc binding domain of SpA. The ligand can be in the form of multimers such as a dimer, a trimer, a tetramer, pentamer, hexamer, heptamer, octamer or a nonamer, which have higher alkali stability than monomers. All units in the multimer can b identical or it can be a heteromultimer, where at least one unit differs form teh others. The polypeptides can be liniked to each other direclty by peptide bonds between the C-temrinal and N-temrinal ends of the polypepitdes. 

Qian (US 2016/0237124) discloses a multimer of a protein A mutant that includes at least two protein A mutants wherein the at least two protein A mutants includes the same amino acid sequence or different amino acid sequences. The multimer can be a dimer, trimer or tetramer. The one or more protein A mutatns are linked together via one or more linker comrpsing 4-10 amino acids. 

Linkage of Domain C units from SpA:

Hall (WO 2008/039141) discloses multimeric chromatography ligands from Domain C from SpA or a functional fragment or variant therof comprised of at least two Domain C nits. In one emboidment, the multimer is comprised of 2-8 units, such as 4-6 units. Linkers may be inserted between the multimer units. In one embodiment, the chromatogrpahy ligand includes no other SpA domains than Domain C. 

Lenght of Linker:

Honda (US 2016/0280744) also discloses domains dervied from protein G or protein A linking the plural domains having affinity for the protein comprising the Fc region of IgG. A polypeptide linker consisting of amino acids is preferable. The number of domains may be optionally selected depending on the use of the protein and the kinds of the domains. The protein can be a dimer, trimer, tetramer or pentamer. The linker has an extended lenght of 8-240 angstrom. The number of the amino acids constituting the peptide linkers is usually about 22-26, preferably about 24-60, dpending on the kind of the amino acids. For example, the peptide linker is preferably 4-10 times repeats of the unit of GlyGlySerGlyGlySer, or mroe preferably 6-10 times rpeats of the same unit. 

Particular Types of Linkers/Spacers:

Zong (US 16,485,854, published as US 2019/0375785) discloses multimers such as tetramers and pentamers of Protein A domains and the Z domain which are connected in series with a spacer. the spacer domain includes an alpha helix or a helix bundel. The alpha helix can contain about 8-32 amino cids, which can form about 2-8 helical turns and ranges from about 10-45A in lenght. The spacer domain can also be a domain of glucagon, a connecting helix of calmodulin, or a single alpha-helix domain. In some embodiments, the spacer is a small protein such as a Sumo domain or an EGF domain. It was discovered that each IgG binding domain of SpA may have a secondary minor Fc binding site in addition to the well-known major Fc binding site. The secondary interaction occurs when the adjacent immunoglobulin binding domain binds to the dominant Fc site. The major and secondary interactions contribue to an high affinity between SpA and IgG due to cooperative effects. In order to eliminate the secondary binding, the neighboring immunoglobulin binding domains can be separated by a spacer. domain. Zong exemplofies a helical domain spacer from the central segment of the connecting helix in ribosomal protein L9 from B. sterothermphilus. The L9 connecitng helix, which is conserved in a number of organissms, was shwon to be exceptionally stable. It has bout 18 residues (forming aobut 4 helical turns and having roughly the same lenght as the Z domain). This tetraZ-H had significantly higher elution pH than Tetra Z connected in eries without the spacers. 

Particular Substitutions

Position 23:

Johansson (US14/358821, published as US 2014/0329995) discloses six or more doamins of protein Z or the C domain of protein A. In one embodiment, the amino acid residue at position 23 is a threonine in all domains of the polypeptide. The mutation improves alkali stability. The domains are liniked by linkers which include up to about 15 mino acid reisudes. In certain emobdiment, the polypeptide is coupled to the solid support by single point attachment such as via thioether bonds.

–position 23 with other substitutions:

Bjorkman (US2013/0274451) teaches multimers of the various domains of protein A with at least one of the monomers having a substitution of the Asparagine at N28 of the B domain or Z domain which results in increase in elution pH. In one embodiment, the asparagine residue at position has also been mutated such as to a threonine.

Position 29:

Bian (US 12/653888 and EP2202310) discloses chromatography ligands comprising 2 or more B or Z domains of SpA attached to a chromatography resin at more than one site on the resin. In some embodiments, the domains differ from the parent amino acid sequence such as at position 29 which is replaced by an amino acid residue other than alanine or tryptophan.

Bjorkman (US 2013/0274451) discloses a ligand form SpA that is made of multimer copies of domain C, with Arginine residue at N28 mutated and optionally, the ligand also contains a G29A mutation.

–With deletion of AAs at N-terminal:

Spector (US9,018,305; US9,234,010; US8,754,196; 8,754,196 and US8,895,706; See also EP2157099)  teaches an affinity chromatogrpahy ligand attached to a solid support where the ligand is based on two or more B domains or two or more Z domains or two or more C domains os SpA, each domain having a deletion of at least 4 consecutive amino acids from the N-terminus starting at position 1 corresponding to the sild type domains and further having a mutation to reduce Fab binding such as a mutation at position 29.

Particular Insertions

Between Positions 3/4:

Yasuoka (US 15/561, 332, published as US 2018/0105560) discloses inserting at least one amino acid between the positions corresponding to positions 3 and 4 of the B, Z or C domain. This is particularly useful when suing repeated domains because steric hindrance causes a problem in producign an affinity chromatography carreir that employs a repeated strucure of the B, C or Z domain. 

See also structure of the SpA (i.e., various domains)    See also Multimers of SpA

One of the problems with proteinaceous affinity ligands in large scale purification is their sensitivity to alkaline conditions. For this reasons, various protein engineering strategies have been used to improve upon affinity ligands. Uhlen (EP1123389), for example, discloses affinity separation where the asparagine (Asn) residues within the affinity ligand are deleted or replaced with a less alkaline sensitive amino acid.

Criticality of binding sites when making Variants

Using phage display technology, researchers modified the IgG binding Z domain into an IgA binding peptide designated as Affibody IgA1 or modified Z domain. This peptide binds both human IgA1 and IgA2. The original IgG binding affinity was completely lost with these modifications (WO 2007/019376).

The repetitive structure of the native SPA gene makes site specific mutagenesis technically diffiult. After mutations of one of the repeats the mismatch primer will, in the next mutagenesis step, anneal more efficiently to a mutated that to a non-mutated repeate. In addition, multiple region mutants are hard to select from a single region mutant by hydridization. These problems severely limit the value of protein engineering in the study of protein A. (Nisson, “A synthetic IgG-binding domain based on staphylococcal protin A” Protein Engineering, 1(2), 1987, 107-13)

Variants, in general

Silverman (US2006/0205016) discloses Staphylococcal protein A variants for binding Ig comprising a polypeptide which varies by one or more amino acids form the sequence of a natural variable heavy chain III (VH3) Ig-fab binding region of spA.

Ljungqvist (WO/2000/063243) describes modified polypeptides which are derivatives of the B domain or Z domain from staphylococcal protein A where between 1 and 20 amino acid residues of said B or Z domain have been substituted by other amino acid reisudes which results in interaction capacity of said polypeptide with at least one domain of human factor VIII protein.

Truncated versions/deltions from Amino or carboxyl ends:

–truncated Zvar/C domain:

Spector (US 8,895,706) discloses an alkaline stable affinity chromatography ligand which includes C domains of SpA with each domain including a G29K mutation as well as 4 amino acids deted from the N-terminus starting at position 1. 

Hansson (US 16/095,721, published as US 2020/0239514) discloses a Zvar and C-domain without the linker region amino acids 1-8 and 56-58. 

Rodrigo (US 10501557) discloses an SpA domain (E, D, A, B, C, Z and  Zvar (without linker region amino acids 1-6) where at least the asparagine or serine residue at position 11 has been mutated to an amino acid selected form the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalamine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

–Truncated version of X domain: (Peyser, WO/2008/127457) disclsoes truncated versions of protein A that include some partion but not all of the X domain of native protein A, do not include a signal sequence and bind specifically to an Fc region of IgG. The versions have the advantage of that it contains some portion of the X domain, which portion significnatly improves its ability to be immobilized for use as an affinity chromatography reagent.

Particular Amino Acids Substituted 

Asparagine (N) residues: See also particular positions below

While SPA is considered relatively stable to alkaline treatment, it has also been though beneficial to improve the stability so that it can withstand even longer exposure to harsh conditions associated with cleaning in place procedures. For this purpose, replacing the asparagine residues has been employed.

Braisted (US6013763) describe Z domain variants of staphylococcal protein A which have significantly reduced size but possess IgG binding affinity equivalent to the wild type Z domain.

Gulich (J. Biotechnology 80 (2000), 169-178) report an SpA where all four asparagine residues replaced by leucine (one residue), aspartate (two residues) and lysine (one resiude). 

Hober (WO 03/080655) teaches an immunoglobulin binding protein such as Staphylococcal protein A (SpA) derivative/analogue wherein at least one asparagine residue of the B-domain and protein Z has been mutated to amino acids other than glutamine or aspartic acid (i.e., such as lysine and leucine) resulting in a ligand with higher binding capacity under alkaline conditions. 

Johansson (WO2010/080065) also teaches ligands of a dimer of domain B of SpA or protein Z wherei the ligands are alkali-stable by mutating at least one asparagine residue to an amino acid other than glutamine. 

Li (US 2014/0031522) discloses an immunogloublin binding proteins with alkali-resistance which includes at least one asparagine residue substituted with a histidine, a serine, an aspartic acid or a threonine residue. 

Shun-Cheng (WO2012/083425 and US2014/0031522) discloses variants of the Z domain where at least one asparagine residue is substituted with a histidine, a serine, an aspartic acid or a threonine reisude. The substitution may confer to the variant an increased stability in alkaline solutions when compared to the wil type.

Uhlen (US6,831,161 and WO 00/23580) also discloses that modification of Asn residues within a protein molecule increases stability of the protein in alkaline conditions and that suitable scaffold proteins include Staphylococcal protein A (SPA) analogue Z (the “Z domain” being a derivative of the B domain of SPA) or other domains or analogues of SPA. 

Lysine Residues:

Hyogo (US 15/883,569, published as US 2018/0215836) discloses an SpA domain where the number of lysine reisudes toward the N-temrinus such as positions 1-38 is eqqual to or greater than a total number of lysine in position 39 and usbsequent positions. 

Hyogo, (US 15/887,381, published as US 2018/0170973) also disclsoes multimers that include a modified C domain where the Lysine at position 4 or 7 is modified to a different amino aicd). 

Majima (EP1992692A1 and US 12/280221, published as US 2010/0286373)  disclose a modified C-domain of SpA or Z-domain which has improved orientation for maintinating affinity for an immunoglobulin and improved chemical stability under acidic pH conditions because a ratio of the number of lysine at positions 39 onwards to the number of lysine at positions 1-38 is increased as compared to a ratio in the unmofided molecule and/or substitution of other amino acid for lysine originally present at positions 4, 7, and 35. 

Majima (US 14/916,316, published as US 2016/0215027) also teaches a multimeric immunoglobulin binding protein having 6-10 total domains from SpA such as the C domain having the general structure (R1)n-(R2m) or (R2m-(R1)n where the R1 domain is an amino acid sequence in which a non-lysine amino acid has been replaced such as the lysine being substituted with a non-lysine amino acid at 1-3 of positions 4, 7, and 35 R2 is an immunoglbulin binding domain coccuring at the N-temrinus or the C-terminus of the protein and includes an amino acid residue that covalently bonds to an insoluble support and the R2 domain is based on an amino acid sequence in which the lysine residue(s) are substituted with a non-lysine amino acid only at position 35, or at posiiton 35 and one or more positions 4, 7 and 35. In one embbodiment the R2 domain includes substitutions of 1-6 amino acid reisdues with lysine at positions 40, 43, 46, 53, 54, and 56. Because only the (R2) domains are selectively immobilized on the support via a covalent bond, it is possible to acheive a highly selective immobilization reaction trhough for example a lysine or a cysteine residue. The R1 in trun has an amino acid sequence that does not contain an amino acid that is active to the chemical reaction used for immobilization. When the immobilization reaction used to immobilzie the prtoien on the support takes place via an amino group, an R2 domain can be produce by substituting the lysine residues contained in the amino acid sequence with non-lysine aino acids only in lysine residues occurring at positiosn that interfere with bidnign to an immunoglobulin upon immobilizing the protein on the support and by substituting some of the non-lysine amino acids not involved in bidning to an immunoglobulin with lysine. In the case of immobilization via a thiol group, a new cysteine residue can be added to the R2 domain. A support immobilization reaction using a disulfide bond or a maleimide group that is highly selective to the thiol group may be used for said immobilization. 

Shima (EP1992692A1) also discloses modified protein of a C domain of Staphylococcus protein A or a Z domain which has improved affinity for an immunoglobulin and also improved chemical stability under acidic pH conditions as compared to an unmodified molecule because the number of lysines at positions 1-38 is increased as compared to the unmodified molecule.

Yoshida (US2014/0107315) discloses E, D, A, B and C domains where all lysine residues are replaced with other amino acids and adding lysine at a terminal. In an alternative embodiment, the lysines at positions 29, 33, 36 and 37 of the C domain is replaced with another amino acid. 

–Gln or Lys to Ala, Ser or Thr:

Hyogo (US 15/876615, published as US 2018/0215785) discloses substitution of a Glutamine (Gln)  or lysine (Lys) in the Fc binding site of an ABP to Ala, Ser or Thr such that the ligand has a lower antibody binding capacity in an acidic pH range. In one e;mbodiment, the substiutions in the C domain are Q9A/G29A, Q9S/G29A, Q9T/G29A and G29A/Q32A.

Methionine and Asparagine-glycine substitutions: 

The methionine residues present in the domains E, D and A make protein A sensitive to cyanogen bromide treatment. In addition, the asparagine-glycine sequences present in all domains makes the protein sensitive to hydroxylamine treatment. Nilsson designed a synthetic IgG binding fragment lacking both the methionine residue and the asparagine-glycine sequence. The synthetic fragment was based on the sequence of domain B, which lacks methionine residues and is thus cyanogen bromide resistant. Finally, a non-palindromic AccI was ntroduced in the 5 end of the fragment to enable polymerization of the fragment. This site introduces a valine residue instead of an alanine residue in position 1. The AccI site is also present in the 3 end of the synthesized Z fragment, thus encoding two additional amino acid residues.  Nilsson “A synthetic IgG-binding domain based on staphylococcal protein A” Protein Engineering, 107-13, 1987).  See also Abrahmsen (US 5,143,844 and EP0230869) for the patent covering this invention (discloses a recombinant DNA fragment Z coding for an immunoglobuilin G binding domain related to SpA where the methionine codon has been replaced with a different amino acid such as valine codon to enable expression of a Met free protein; also discloses G-A substitution).

Proline residues:

Bjorkman (WO 2012/074463) discloses one or mroe of a protein A domain (E, D, A, B, C) or protein Z or a functional variant thereof with at least one of the monomers having a substitution of the C-temrinal most proteline residue after teh third alpha-ehlix. 

Hydrophobic Amino Acids for other Hydrophobic or polar uncharged amino acids

Hyogo (US 15/876597, published as 2018/0215795) discloses engineered protein A ligands dervied from one of the E, D, A, B and C domains in which a hydrophobic amino acid residue in an Fc binding site (e.g., Phe at position 5 or 13, Leu at position 17 or Ile at position 31) is substituted by a different hydrophobic amino acid (Gly, Ala, Val, Leu, Ile, Met, Phe, or Trp) or polar uncharged amino acid residue (Ser, Thr, Gln, Asn, Tyr, and Cys) where the protein has a reduced antibody-binding capacity in an acidic pH range as compared to teh wild type protein. 

Affinity tags and Coupling elements (cysteine, Histidine , arginine, lysine) 

Protein A/G with Basic amino acid (e.g., histidine) tags:  See also modified Z domains with Histidine linkers below

Katayose (US2010/0105879A1) discloses immobilization of a protein having affinity for an antibody molecule such as Protein A/G having a tag sequencefrom preferably 5-30 amino acids  that includes a basic amino acid such as a histidine to a support which has at least one functional group such as carboxyl, epoxy or tosyl group.

Ljungquist (Eur. J. Biochem. 186, 557-561 (1989) discloses introduction of a cysteine into the C temrinal part of SpA, containing one, two or 5 IgG binding domains and subsequently immobilizing the domains on thiopropyl-Sepharose. The gel was then used for affinity purificaiton of human IgG. 

Rodrigo (Wo2106/079033) eaches protein A mutants which include coupling elements such as cysteine residues, a pluarlity of lysine resiudes and a plurality of histidine residues. The coupling elements can be located within 1-5 amino acid residues from teh C or N terminal end. 

Wagner (US6,406,921B1) disclose immobilization of protein via an affinity tag which may comprise a poly-cysteine, poly-lysine, poly-arginine or poly-histidine. The position of the amino acid tag can be at the amino or carboxy-terminus of the protein. An adaptor molecule such as protein A or G that links the affinity tag to the immobilized protein can also be used.

Histidine Linker-Z domain: 

Anderson (US2011/0136169A1) teaches a vector with N- or C terminal His tags fused to the upstream synthetic protein A IgG binding (“Z”) domains.  Separating the ZZ domains from the cloning site is an intervening linker region containing coding sequences for a tobacco etch virus (TEV) protease cleavage recogniction site which allows cleavage and release of the cloned target. Targets fused to His-Tags (at the C terminus) can thus be purified by metal affinity chromatography.

Chen, (“Immobilized protein ZZ, an affinity tool for immunoglobulin isolation and immunological experimentation” Biottechnol. Appl. Biochem. (2006) 45, 87-92) discloses recombinant ZZ protein with a hexahistidine tag at the N-terminus which exhibited good performance in affinity purification of IgG.

Tamori (US13/36410) discloses the formula R-R2 where R represents an amino acid sequence consisting of 4-300 amino acid residues containing a region of 4-20 contiguous histidine residues (also referred to as a “histidine linker”) and R2 represents an amino acid sequence consisting of 40-500 amino acid residues containing Z domain of Protein A. The ligand is immobized on a carrier and showed excellent slakli resistance. It is thought that when a regio of continguous histidine residues is added to the Z domain, the position of binding between the carrier and the Z domain leads to a structural change leading to an increase of alkli resistance. 

Particular Amino Acids Inserted or Deleted

Acidic amino acids (e.g., aspartic acid or glutamic acid):

Yoshida (US 14/916263, published as WO/2015/034056) discloses increasing the number of acidic amino acids in the C or Z domains of Protein A. Acidic amino acids include an amino acid having a carboxyl group in a side chain such as aspartic acid or glutamic acid. In one embodiment, the amino acid substituted by the acidic amino acid is at least one at positions 7, 18, 40, 46 or 55. 

Histidine amino acids:

Masaya (JP2010081866) discloses an improved protein A that is obtained by replacing an amino acid resiude having an exposed surface area ration of greater or equal 35% with a histidine residue. 

Shinya (US 9382297 and EP 2728000) discloses a protein A mutant protein having reduced affinity in acidic region where any one of Phe5, Asn6 Gln9, Gln10, Asn11, Phe13, Tyr14, Glu15, Leu17, Glu24, Glu25, Arg27, Asn28, Ile31, Gln 32, Lys35 and Asp36 are substiuted by a histidine residue. 

amino acids added to either end: Martin (EP2066419) discloses a modified Spa Domain C where the Asn-Lys_Phe-Asn in positions 3-6 are deleted or where one or more amino acids are added to either end of the WT Domain C. In some embodiments, the addition comprises the mutation G29A.

Spector (US8,754,196 and US 8, 895, 706) teaches a modified SpA ligand from the B, D and Z domains where at least 3 consecutive amino aicds form the N-terminus are deleted. 

Particular Positions (by type of amino acid (1 letter code)):

D36:

Knick (US 2019/0300568) discloses Fc binding prtoeins which remainstable even after akaline treatment. In one emdobiment, the Fc binding protein includes 2-6 Fc binding domains linked to each other. In one obdiment, the ligand include D36H. 

Tsukamoto “Engineered protein A ligands, derived form a histidne-scanning library, facilitate the affinity purificaiton of IgG under mild acidc conditions” J Biological Engineering, 2014, 8: 15) disclsoes a D36H mutation whose binding of IgG was highly senstive to pH change. The antibodies could be leuted at a higher pH when used as a ligand. 

E24: Bian (US 12/653888) discloses generation of an SpA B domain variants with the glutamic acid at position 24 mutated to all other naturally occurring amino acids except cysteine (C), serine  (S), alanine (A), glycine (g), asparagine (N), and glutamine (Q).

F5 or F13: Hyogo (US 15/876597, published as 2018/0215795) discloses substitution of the Phe at a position 5 or 13 of the C domain C, B, Z, E, D or A domains with a different hydrophobic amino aicd such as Gly, Ala, Val, Leu, Ile, Met, Phe, or Trup or by a polar uncharged amino acid such as Ser, Thr, Gln, Asn, Tyr or Cys. 

G29A:

Hall (US 9290549) disclsoes one or more Domain C units form SpA with the mutation G29A which after 5 hours incubation in 0.5 M NaOH has retained at least 95% of its orignal binding capacity. 

Q9 Q32 or Q40 or Q55: Rogrigo (US 2016/0159857 and WO 2015/005859) disclsoes an Fc binding polypeptide with improved alkaline stability such as the B or C domain of SpA where the glutamine residue at position 9 has been substituted with an amino acid other than asparagine, proline or cysteine, such  as to a tryptophan, leucine, glutamic acid, valine or lysine. This has the advantage that deamidation sensitive asparagines are not introduced, that the chain cofrmation is not disturbed by introduction of prolines and sites for disulfide bridges are not introduced. In another embodiment the glutamine 32 has been mutated to an amino acid other than asparagine, glutamine, proline or cysteins, such as an alanine.  In some embodiments, the glutamine residue at position 40 is the one mutated.

Hyogo (US 15/876,615, published as US 2018/0215785) disclsoes methods for purifying antibodies using a ligand from the D, A, B and C domains of Protein A where the Gln at positions 9, 10 and 32 of for example the C domain has been mutated to Ala, Ser, or Thr. 

L17: Hyogo (US 15/876597, published as 2018/0215795) discloses substitution of the Leu at a position 17 of the C domain C, B, Z, E, D or A domains with a different hydrophobic amino aicd such as Gly, Ala, Val, Leu, Ile, Met, Phe, or Trup or by a polar uncharged amino acid such as Ser, Thr, Gln, Asn, Tyr or Cys.

H18:  

Ander (US14/385,336) discloses an immunoglobulin or Fc binidng protein that has one or more domains of Spa A (i.e., the E, D, A B and/or C domains) or protein Z domain where the Asparagine or Histidine at the position corresponding to H18 of the B or Z domain has been substituted with an amino acid which is not proline or Asparagine. Further, if the amino acid at position corresponding to P57 of the B or Z domain is Proline and the amino acid residue at position N28 of the B or Z domain is Asparagine, then the aino acid at position H18 of the B or Z domain is not Serine, Threonine or Lysine. An advantage of the mtuated protein is that it binds Fc containing proteins and rleases them at pH levels higher than Fc binding proteins not have the H18 mutation. 

I31: Hyogo (US 15/876597, published as 2018/0215795) discloses substitution of the Ile31 at a position 17 of the C domain C, B, Z, E, D or A domains with a different hydrophobic amino acid such as Gly, Ala, Val, Leu, Ile, Met, Phe, or Trup or by a polar uncharged amino acid such as Ser, Thr, Gln, Asn, Tyr or Cys.

N11 (or N3 in truncated Z or C domain):

Rodrigo (US 10501557) discloses an SpA domain (E, D, A, B, C, Z and  Zvar (without linker region amino acids 1-6) a where at least the asparagine or serine residue at position 11 has been mutated to an amino acid selected form the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalamine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Rogdrigo (US 15/282367, published as US 20170327534) discloses SpA analogues where at least the asparagine or serine in the case of Domain D of SpA has residue at position 11 in the B, C or Z domain has been mutated to an amino acid such as glutamic acid, lysine, tyrosine, threonine, phenylalamine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine. The mutation of N11 in these domains confers an improved alkali stability. 

Rodrigo/Hansson (US 16/095721, published as US 2020/0239517) see also US 10703774) discloses a Zvar and C-domain without the linker region amino acids 1-8 and 56-58 where the amino acid residues at positions 13 and 44 are asparagine and wherein at least hte asparagine residue at position 3 (corresponds to position 11  for the B, C or Z domains) has been mutated to an amino acid selected form the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine alanine, histidine and argineine. In one embodiment the matrix is provided in a stroage liquid that includes at least 50% by volume of an aqueous alkali metal hydroxide solution and storing the matrix for at least 5 days. 

N6 or 11:

Hober (US 20160152668) discloses a plurality of lgiands coupled to a solid support wherein at least hte asparagine residue at position 6 or 11 or a parental immunoglublin binding protein has been mutated to an amino acid other than glutamine which confers increased chemical stability at alkaline pH. 

N23T:

Johansson (US 20140329995) disclsoes multimers of Protein Z or C domain of Protein A such as the Z prteoin with an N23 mutation. 

Hober (US 7834158, US 8198404, US 8354510, US 9296791, US 9534023, US 10918971 and US 9156892) disclsoes a modified B or Z domain where one asparagine residue has been mtuated with an amino acid other than glutamine. In advantageous embodiments, the mutations are N23T N23T and N43E, N23A, N6A, N11S, N11S and N23% and N6A and N23%. The mutations(s) confer an increased chemical stability at alkaline pH value.

Linhult (“Improving the tolerance of a Protein A analogue to repeated alkaline exposures using a bypass mutagenesis approach, Proteins 55: 2004, 407-416) discloses an engineered SPA Z form where the asparagine 23 is exchanged for a threonine, thereby increaseing the stability to alkaline treatment compared to the native Z molecule.

N28X:

Bjorkman (US13/996023 & WO2013147691) teaches modified SpaA B and Z domains where the Asparagine at position 28 has been substituted with another amino acid (i.e., N28X).

Jendeberg (J. Molecular Recognition, 8, 270-278 (1995) dicloses mutant Z proteins with an N28A substitution.

G29A:

–Z domain: (A1V/removal of Methionine & G29A): 

Protein Z is a synthetic construct derived from the B domain of SpA, wherein the glycine in position 29 has been exchanged for alanine (Ander, US 14,385, 336, citing Stahl et al., 1999). 

The Z domain of SpA is actually an engineered analogue of the B domain of SpA and includes an alanine instead of glycine residue at position 29 (G29A). The 1st amino acid ala is also replaced by Val (A1V).  Thus the Z-domain is a synthetic IgG binding protein, consisting of 58 amino acids, which are derived from the B domain of staphylococcal Protein A. It is designed to lack the Asp-Gly dipeptide sequence as well as methionine residues, so it is resistant to treatment with hydroxylamine and CNBr, in contrast with native Protein A (Chen, “Immobilized protein ZZ, an affinity tool for immunoglobulin isolation and immunological experimentation” (Biottechnol. Appl. Biochem. (2006) 45, 87-92). The Z domain binds to the Fc region of immunoglobulins as do the 5 homologous SPA domains, but unlike the parental domain it does not bind to the Fab region. The term “affibody” is used to define a class of engineered proteins selected fro their specific binding activity towards a desired target and based on the Z domain.

In the Z domain, another mutation is simultaneously introduced to substitute Ala at position 1 of the B domain by Val. This mutation is intedended to facilitate the genetic engineering preparation of a gene encocoding multiple domains linked together and does not affect the domain functions. (WO 2012/165544)

Nilsson showed that the asparagine side chain of SPA forms a hydrogen bond to the side chain of a glutamine in the Fc fragment which suggested that substitution of this asparagine residue in domain B would be likely to affect the IgG interaction. In contrast, the neighboring glycine residue (Gly 29), unusually placed in an alpha helix, did not seemed to be involved in the IgG interaction. Thus a methyl group was added to the glycine converting it into an alanine. The substitution was found to be sterically aceptable to the structure. This “Z domain” thus lacked methionine residues being based on fragment B and the glycine residue at position 29 had been substituted by an alanine to change the asparagine-glycine sequence. Finally, a non-palindromic AccI site had been introduced in the 5 end of the fragment to enable polymerization of the fragment. This site introduces a valine residue instead of an alanine residue in position 1. The AccI site is also present in the 3 end of the synthesized Z fragment, thus encoding two additional amino acid residues.  Nilsson “A synthetic IgG-binding domain based on staphylococcal protein A” Protein Engineering, 107-13, 1987).  See also Abrahmsen (US 5,143,844 and EP0230869) for the patent covering this invention (discloses a recombinant DNA fragment Z coding for an immunoglobuilin G binding domain related to SpA where the methionine codon has been replaced with a different amino acid such as valine codon to enable expression of a Met free protein; also discloses G-A substitution).

Hober (US2006/0194950) discloses alkali stable domain that can be further modified such that the ligands lack affinity for Fab but retains Fc affinity, for example by a G29A mutation. 

Jansson (FEMS Immunology and Medical Microbiology 20 (1998) 69-78 disclose an engineered domain Z, similar to fragment B but with a single glycine to alanine amino acid substitution.

Bjorkman (US 2013/0274451) discloses a ligand form SpA that is made of multimer copies of domain C, with Arginine residue at N28 mutated and optionally, the ligand also contains a G29A mutation.

–Replacement of G29 with amino acid other than alanine or tryptophan:

Spector (EP2157099A1; US8,592,555, US2010/0063256) discloses modified immunoglobulin binding proteins such as SpA based on one or more domains of SpA (i.e., E, D, A, B, C and Z) which are modified to replace at least the amino acid at position 29. In the case of the domains E, D, A, B and C, the amino acid at position 29 is a glycine, which is replaced with an amino acid other than alanine or tryptophan. In the case of domain Z, the amino acid at position 29 is an alanine, which is replaced with an amino acid other than glcine or tryptophan. Spector (US8,592,555) discloses a modified C domain of SpA where the glycine residue at position 29 is replaced with a lysine amino acid residue such that the SpA binds an Fc portion of an immunoglbulin but exhibits reduced binding to a Fab portion of an immunoglobulin. Spector (US 14/061,080, published as US 2014/0046037 teaches B and C domains where the glycine (G) at position 29 is substituted with lysin (K), Leucine (L) or arginine (R) and where the alanine (A) at position 29 of the Z domain is substituted with lysine (K), Leucine (L) or arginine (R). 

Q9:

Rodrigo (US 20160159855, 20160159857) disclsoes a mtutant B or C domain of SpA wherein at least the glutamine at position 9 has been mutated to an amino acid other than asparagine. 

V40 (C domain): Hyogo discloses a C domain ligand for affinity chromatography where the valine is exchange for a polar uncharged amino acid such as a thr, Ser, Gln, Asn or Cys or a basic amino acid such as a his, lys or Arg. 

Methods of Generating

Align sequences of domains (in silico) – statistically fragment (in silico) –reassemble so order remains is maintained

Ulrich (US 15/744,147, published as US 2018/0305463)discloses a method of generating a non-natural Ig binding protein from known sequences of protein A domains (E, D, A, B and C) by alignment of the known sequences, statistical fragmentation in silico (performed on a computer) , and then in silico assembly of new sequences from the various fragments such that the relative order of the statistically fragmented sequences in the amino acid sequence of the non-natural immunoglobulin binding protein is maintained as said statistically fragmented sequences present in the amino acid sequences of Ig binding domains of the Protein A polypeptides. 

Mutate domain (B2 domain of protein G)  –Express gene sequences –Purify by affinity chromatography –Mass Spectromety

Honda (US 2015/0183820) discloses a method of generating a variant Protein G which includes the steps of selection of the target part for teh mutation based on an alysis of teh surface bound to the Fc region (3D coodinate dats of the complex of the B2 domain of the protein G and the Fc region of Ig1 downloaded from Protein Data Bank (PDB) and amino acdi residues of the B2 domain within a set angstrom distance from teh Fc region with an exposed surface area ratio was selected as the target parts for the mutation), disgning the base sequences of the genes encoding the variant proting G (using Gene Designer), synthezing plasmid vectors which contain the genes encoding the variant protin G, transformation of E coli with the vectors, purifying the variants with affinity chromatography (IgG Sepharose  Fast flow column), measuring MW of the improved protein G with MALDI-TOF type MS and comparing MW of a peak detected form the MS and theoretial MS calculated form teh amino acid sequence of the produced mutant prtoin to oconfirm target protein. 

As to purification of bispecific antibodies based on altering amino acids in the antibody see purificaiton of bispecific antibodies 

Structural Comparison between the SpA Domains

It is known that Protein A is comprised of domains called E, D, A, B and C each of which has about 60 amino acids and these domains show high homology to each other and can bind to the common Fc region of immunoglobulin independently. (Sato, US2010/0168395).

Domains A, B and C of spA are highly homologous, with no amino acid differences within the two alpha-helices. Domain D is slightly more diveresed with three amino acid substitutions in the alpha helical regions. Region E is strikingly different, in particular within the N-temrinal segment. The first eight residues have been substituted by six others. Despite these differences region E binds IgG. (Nilsson, Protein Engineering, 192), pp. 107-113 1987).

Organization of the Protein A gene. S=signal sequence, A-E=IgG binding domains and X=C-terminal with no IgG binding activity.

From US12/653888.  AA alignments of the wild type IgG binding domains of SpA domains (E, D, A, B and C).

Staphylococcus aureus (“SpA”) refers to a 42 Kda multi-domain protein isolated from the bacteriumStaphylococcus aureus. SpA is bound to the bacterial cells wall via its carboxy-terminal cell wall binding region, referred to as the X domain. At the amino-terminal region, it includes 5 immunoglobulin binding domains, referred to as E, D, A, B, and C. Each of these domains contains about 58 amino acid residues, and they share 65-90% amino acid sequence identity. 

Alignment of common Wild-type (WT) SpA domain sequences (sequences taken from Bian, 2010/0221844) 

--B/C

B domain: ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

C domain: ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK

 91.4% identity in 58 residues overlap; Score: 272.0; Gap frequency: 0.0%

 1 ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

1 ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK

 
--B/Z

B domain: ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

Z domain: VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

98.2% identity in 57 residues overlap; Score: 288.0; Gap frequency: 0.0%

2 DNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

2 DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

--C/Z

C domain: ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAP

Z domain: VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

89.3% identity in 56 residues overlap; Score: 257.0; Gap frequency: 0.0%

 2 DNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAP

2 DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAP

                 ********************* ***** ********** *   *************

Residues on SpA involved in binding

Protein A (SpA) exists in both secreted and membrane associated forms possesses two distinct Ig binding activities: each of its domains E, D, A, B and C can bind Fcy (the constant region of IgG involved in effector functions) and Fab (the Ig fragment responsible for antigen recognition. (Graille, PNAS, 97(10), May 9, 2000, pp. 5399-5404). 

SPa binds human immunoglobulins at two target sites: one in the Fab portion of the immunoglobulins of the IgM, IgA, IgG and IgE isotypes and the other in the Fc portion of IgG1, IgG2, IgG3 allotype G3m(s,t) and IgG4 immunoglobulins. It was demsontrated that these two SPA binding sites are distinct when chemically modified SPA lost the ability to bind Fcy but retained ability to bind in the Fab porition and by inhibition analysis in which IgG F(ab’)2 fragments but not IgG Fc fragments, blocked SPA binding to IgE. (Potter “Staphylococcal Protein A binding to VH3 encoded immunoglobulins”, Intern. Rev. Immunol., Vol. 14, pp 291-308, 1996). 

While all five domains of Protein A (A, B, C, D, E) bind IgG via their Fc region, only domains D and E exhibit significant VH3 binding. (Slough, US Patent Application No: 15/566231, published as US 2018/010007). 

SpA possesses distinct Ig binding sites. One site is for Fcy (the constant region of IgG class of Ig) and the other is for the Fab portion of certain Ig molecules (the porition of the Ig that is responsible for antigen recognition). The Fcy binding site has been localized to the elbow region at the CH2 and CH3 interface of most IgG subclasses, and this binding property has been extensively used for purification of antibodies. Silverman (US2006/0205016).  

Protein A interacts with antibodies through two distinct binding evests: the “classical” binding site on the Fc portion of human IgG1, IgG2, and IgG4 and the “alternate” binding site found on the Fab portion of human IgG, IgM, IgA and IgE that contain heavy chains of the VH3 subfamily. (Starovasnik, Protein Science, 1999, 8; 1423-1431).

Fcy binding site: 

The Fcy binding site of SpA has been localized to the elbow region at the CH2 and CH3 interfact of most IgG subclasses and this binding property has been extensively used for the labeling and purificaiton of antibodies. (Graille, PNAS, 97(10), May 9, 2000, pp. 5399-5404).

The N-terminal part of the mature protein is responsible for the binding to the Fc-portion of IgG (this is the region that has the 5 hoologous domains each of about 58 amino acid residues which are individually IgG binding). Each of the domains have two alpha-helices involved in the IgG binding. The A, B and C domains are highly homologous, with no amino acid differences within the two alpha-helices. Region D is slighly more diverged with three amino acid substitutiosn in the alpha helical regions. Region E is strikingly different, in particular within the N-terminal segment. (Nilsson, “A synthetic IgG-binding domain based on staphylococcal protin A” Protein Engineering, 1(2), 1987, 107-13).

Protein A binds the Fc region of immunoglobulins. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. It binds with high affinity to human IgG1, IgG2 and IgG4 as well as mouse IgG2a, IgG2b and IgG3. Protein G is recommended for all mouse isotypes  (WO2008/091740). Protein A binds with moderate affinity to human IgD, IgM, IgA and IgE as well as mouse IgG1. One molecule of immunobilized protein A can bind at least two molecules of IgG. The advantage of Protein A compared to Protein G is that the binding of IgG is weaker and consequently milder conditions can be used to release IgG from Protein A (WO1997017361).

–Residues in the D domain:

Graille, (PNAS, 97(10), May 9, 2000, pp. 5399-5404) disclosed the cyrstal structure of domain D of SpA complexed with the Fab fragment of human IgM and the key contact residues from both partners in the interaction. The residues in domain D involved in the interaction with Fab are highly conserved in other Ig binding domains of SpA and are distinct from the residues that mediate binding to Fcy above. The interaction involves the following domain D residues: Gln26, Gy29, Phe-30, Gln32, Ser88 and Asp 36 of helix II; Asp37 and Gln 40 in the loop between helix II and helix III; and Asn43, Glu47 and Leu51 of helix III. In the Fab, the interaction is mediated by heavy chain residues GlyH15, SerH17 in the beta turn befroe strnd B; Arg H19 of strand B; LysH57 and Tyr H59 of strand C; lys-H64, Ly-H65 and Arg-H66 before strand D, Thr-H68 and Ser-H70 of strand D, Gln-H81 of strand E; Asn-H82a and Ser-H82b after strand E. Six of these residues are in framework region (FR) beta strands whereas the other seven residues are in VH region FR interstrand loops on the side farthest from the antigen binding pocket. 

Silverman (US2006/0205016) teaches that the Z domain binds to the Fc region of immunoglobulins as do the 5 homlogous SpA domains, but unlike the parental domain it does not bind to the Fab region (¶102).

Residues on the Antibody Important for Binding

Protein A binds to the framework regions of V domains of the human VH3 family and to certain V domains of the VH1 family. (Holliger, US 5,837,242).

SpA, which exists in both secreted and membrane-associated forms, possesses two distinct Ig-binding adtivities: each domain can bind Fcy (the constant region of IgG involved in effector functions) and Fab (the Ig fragment responsible for antigen recognition). SPA is a 42-kDa protein covalently anchored to the staphylococcal cell wall through its carboxyl temrinal end. The protein is comprised of five repeated domains (E, D, A, B, C) of 48 reisudes linked to teh cell surface by region Xr, which contains a variable number of short 8-residue repeats. Each SpA domain can bind with high affinity to the Fc region of IgG and to teh Fab region of immunoglobulin of the VH3 subclass. (Lydon, US 2016/0068589). 

Fab binding site:

A subset of antibodies bind Protein A in the variable region of the heavy chain. In particular, IgGs that contain heavy chains dervied from the H3 gene fmaily have been shown to exhibit this behavior. Nearly half of human VH germline genes belong to this family. Becasue of this, on SpA-based resins, many bsAbs exhibit poor resolution of the two binding species as well as retention of the non-binindg Fc&Fc& parental antibody. This is becasue VH binding reduces the avidity differences between the bispecific and the FcFc parental and Fc*Fc* parental is also retained by VH binding. This phenomenon can be resolved through the use of the alkli stablized MabSelect SuRe ligand. This is becasue binding binding studes between SpA and antibodies have shown that all five domains of SpA (E, D, A, B, and C) have antibodies via the Fc region, only domains D and E exhibit significant Fab binding. As the MabSelect SuRE lgiand is a treamer of the Z-domain, a protein-engineered version of the native SpA B domain, it would be expected to lack significnat Fab binding, and this has been demonstrated in multiple studies. (Tustian “Development of a novel affinity chromatography resin for platform purificaiton of bispecific antibodies with modified protein A binding avidity, 2018). 

–Framework Subgroup 3 (VH3) domain encoded antibodies:

The term “VH3 domain” refers to the framework subgroup 3 of human heavy chain variable region of an immunoglobulin. The heavy chain variable domains of antibodies are classified into distinct subfamilies (VH1 to VH6) on the basis of DNA sequence and protein homologies. (Slough, US Patent Application No: 15/566231, published as US 2018/010007)

The Fab specificity of SpA is less well characterized but it has been shown to involve a site on the variable region of the Ig heavy chain. Fab binding specificity is restricted to products of the human variable region of the Fab heavy chain VH3 family that represent nearly half of inherited VH genes and their homologues in other mammalian species. Graille, (PNAS, 97(10), May 9, 2000, pp. 5399-5404) In the context of IgG purification using Protein A, VH3 domain interactions have been considered undersirable given that they affect the elution profile of the antibody to be purified and alternative resins have been developed that contain only domain B of Protein such as Sure from GE Healthcare. However, many of the antibodies and antibody derived molecules currently available and in development such as antibody fragments like Fab do not contain Fc regions and binding of protein A by VH3 regions enables the use of protein A in the purification of these types of antibodies. (Slough, US Patent Application No: 15/566231, published as US 2018/010007).

An alternate binding site for binding protein A has been described in antibodies that contain the specific framework subgroup 3 of the human H chain variable region, also referred to as the VH3 domain and has often been classified as a secondary interaction.  (Sasso, J. Immunol 1991, 147: 1877-1883, Human IgA and IgG F(ab’)2 that bind to staphylococcal protein A belong to the VHIII subgroup). While all five domains of Protein A (A, B, C, D, and E) bind IgG via their Fc-region, only domains D and E exhibit significant VH3 binding. These VH3 domain interactions with Protein A have been considered undesirable given that they affect the elution profile of the antibody to be purified, and alternative resins have been developed and are available on the market that contain only domain B of Protein A such as SuRe® from GE Healthcare. However, many of antibodies in development do not contain Fc regions and binding of protein by VH3 regions enables the use of protein A for the purification of such antibodies. (Slough, US Patent Application No: 15/566231, published as US 2018/010007)

Immunoglobulins of human heavy chain subgroup III have a binding site for SpA on the heavy chain variable domain (VH), in addition to the well known binding site on the Fc portion of the antibody (Starovasnik, Protein Science, 1999, 8; 1423-1431). Protein A shows binding to some human VH domains of the VH3 family but not to VH1 and VH2 (Jansson (1998) FEMS Immunology and Medical Microbiology 20: 69-78). 

SPA binds human immunoglobulins in the variable region encoded only by gene segments belonging only to the VH 4amily. The simultaneous interaction of amino acid resiudes in FR1, CDRH2 and FR3 of VH3 encoded antibodies is required for binding to SPA. When any one of these regions is replaced by sequences from a non-SPA binding antibody, the ability to bind SPA is abrogated. (Potter “Staphylococcal Protein A binding to VH3 encoded immunoglobulins”, Intern. Rev. Immunol., Vol. 14, pp 291-308, 1996). 

Nearly half of human VH germline genes belong to the VH3 subfamily and pharmaceuticals containing IgG antibodies of the VH3 subfamily are under study and some of them are alaredy commercially available. (Yoshida, US 2012/0208234). 

Hillson discloses that the ability of human VH3 Ig to bind SPA via their Fab region is analogous to the binding of bacterial superantigens to T cell receptors. Hillson showed that binding to SPA in ELISA cocurred with 15 of 15 VH3 IgM, but none of 12 IgM from the VH1, VH4, VH5 or VH6 vamilies. Use of D, JH and Cl genes was similar among VH3 and non-VH3 IgM. A comparison of the corresponding VH protein sequences identified a probably site for SPA binding that includes VH3 residues in the framework region 3 (FR3) and perhaps FR1 and 3′ complementary determining region 2. The results thus demonstrate that among human IgM, specificity for SPA is encoded by at least 11 different VH3 germline genes and that SPA likely binds to residues in the VH framework region, outside the classical antigen binding site of the hypervariable loops. (J Exp. Med, 178, 1993, 331-336). 

SpA is a 45 kDa bacterial membrane prtoein that can interact with either Fcy, a constant reigon portion of IGG, or with the Fab portion that also mediates conventional Ag binding. SpA has been shown to specificlally interact with Fab dervied form the VH3 family, and is little aaffected by VH CDR3, JH or light chain usage. The 61 mino acid sequence of SpA that represents domain D exhibited both the FH3 Fab and Fcy binding specificites. It had the strongest binding with an IgM-k encoded by the germline configuation of the VH3 gene VH26C (Roben, “VH3 family antibodies bind domain D of Staphylococacal Protein A” J Immunology, 1995, 154: 6437-6445). 

Jansson (FEMS Immunology and Medical Microbiology 20(1) (1998) 69-78 discloses that all the domains of SpA (A, B, C, E and D) bind Fab with similar strenght, contrary to reported work that only domains E and/or D are responsible for Fab binding. Most amino acids reported to be important for good SPA binding are present in the DP_53 VH3 region. However the Z domain having only a 1 amino acid difference (glycine to alanine mutation at posiiton 29) shows very little or no binding activity. 

Potter, (Intern. Rev. Immunol., 14, pp. 291-308) discloses that replacement of a single involved region with the corresponding region from a nonbinding Ig resutls in loss of ability of VH3 encoded Ab to bind SPA.  SPA binds human immunoglobulins in the variable region encoded only by gene segments belowing to the VH3 family. 

Potter (J. Immunology, 1996, 157: 2982-2988) also disclsoes that at least 15 different VH germline genes have been shown to bind SPA. A single amino acid change at position 57 in the CDR2 of a human SPA nonbinding VH encoded rheumatoid factor converted it to an SPA binding, implicating CDR2 in SPA binding. When regions of the mutated binder were exchange with those form a mouse nonbinding antibody, the pattern of SPA binding indicated that residues in CDR1, CDR2 and FR3 are involved in the interaction between VH3 encoded antibodies and SPA. In addition, all three regions are simultaneously required for SPA binding to occur because when any one of the three regions are altered, SPA binding is disrupted. 

Yeung (WO2010/075548) discloses a variant IgG comprising a human IgGVH region having one or more amino acid substitutions relative to the WT IgGVH region which has altered binding to Staphylococcus aureus prtoein A. In some embodiments, the variant has increased binding to protein A by an amino acid substitution at residue(s) 70, 79, and 82 (e.g., S70A, Y79A or S82bA). In some embodiments, the variant has decreased binding to protein A as by substitution in the IgG VH region at residues 17, 19, 57 66 81 and 82a (e.g., S17A, RI9A, T57A, T57K, R66A, Q81A or N82aA). In some embodiments, the variant IgG is Ig1, IgG2, IgG3 or IgG4. 

–Particular substitutions in VH3 Germline/Subclass region: 

—-T57E:

Bodn (US 2005/0079574) discloses that in classical VHH3 domains, the mutation T57E abolishes affinity for protein. 

Igawa (EP2522724 and WO/2011/078332) disclsoes that the H chain variable region of the VH3 subclass has protein A binding activity and that accordingly to increase protein A binding, the amino acid sequences are preferably identical to those of the H chain variable region of the VH3 subclass and to reduce protein A binding, the amino acid sequences are preferably identical to those of the H chain variable region of another subclass. Such modifications can be part of purifying multimers/bispecific antibodies such that amino acid residues in either or both of the first/second polypeptides are modified so that there is a larger difference of protien A binding ability between the first and second polypeptides. 

As to the different isotypes of IgG

Protein A bind to the CH2-CH3 interface of human IgG1, human IgG2 and Human IgG4 (Fc) and as such cannot be used for the purification of human IgG3. Protein A shows binding to some human VH domains of the VH3 family but not to human VH1 and VH2 and thus Protein A cannot be used for purification of all human IgG derived Fab gragments. In addition A domain based Protein A variants (like MabSelect Sure) lack the ability to bind to human VH3 domains and as such can not be used for Fab purification. (Hermans, 13.982970). 

CH3 domain modifications:

Davis (WO/2010/151792) discloses a bispecific antibody comprising a H chain variable domain that are differentially modified in the CH3 domain wherein the differential modificaitons result in a differential affinity for the bispecific antibody for an affinity reagent such as Protein A. For exaple, the second CH3 region can include a 95R modification(by IMGT exon numbering or 435R by EU numbering). in another embodiment, the second CH3 region includes a 436F by EU modifiecation. In another embodiment,t he second CH3 region is from a modified human IgG1 and fruther includes a modificaiton selected from D16E, L8M, N44S, K52N, V57M and V821. In one emboeidment, the second CH3 region is from a modified human IgG2 and includes a modification selected from N44S, K52N and V82I. In anotehr embodiment, the second CH3 region is from a modified human IgG4 and includes Q15R, N4S, K52N, V57M, R69K, E79Q and V82I. 

Domains of SpA 

D domain is a 61 amino acid polypeptide that folds into a 3 helix bundle structure. It is capable of Fc bnding via residues on the surface of helices 1 and 2, or to Fab via residues on the surface of helices 2 and 3.

Silverman (WO 00/69457) discloses critical residues in domain D that interact with Ig-Fab to mediate the Ig-Fab binding site in SpA. (Roben, J. Immunogloy, 1995, 154: 6437-6445)

Binding studies demonstrate that domain D possesses the same VH3 restricted Fab binding specificity as the native SpA and also possesses Fc-y-binding (Fcy is a constant region porition of IgG) activity. Structurally domain D is in part distinguished form other domains of SpA because it has a three amino insertion between the second and third amino acids in the consensus sequence. 

A domain of Spa is a 58 amino acid polypeptide that also folds into a 3 helix structure. It is capable of Fc binding via residues on the surface of helices 1 and 2, or to Fab via residues on the surface of helices 2 and 3.

B domain is a 58 amino acid polyeptide that also folds into a 3 helix bundle structure. It is capable of Fc binding via residues on the surface of helices 1 and2, or to Fab via residues on the surface of helices 2 and 3.

C domain is a 58 amino acid polypeptide that also folds into a 3 helix structure and capable of Fc binding via residues on the surface of helices 1 and 2 or to Fab via residues on the surface of helices 2 and 3 (Bian US 12/653888). 

The C domain has superior resistance to alkaline conditions compared to the other domains. Thr-23, an amino acid found only in the C domain, is easily predicted to have a beneficial effect on alkaline stability. Val-40 one of the unique residues of the C domain appears to have a positive effect on its alkaline stability. The C domain also lacks a protease susceptible Gly-Glu sequence. Teh G29A mutation in the C domain increases alkaline stability, although smaller than that observed for teh B domain. (Yoshida “Remarkable alkline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution” (2013), Protein Science). 

Hall (WO 2008/039141A1) disclose Domain C from SpA and variants thereof. 

Z domain of SpA (see analogues of SpA) is an engineered analogue of the B domain of SpA and includes an alanine instead of glycine residue at position 29. The Z domain binds to the Fc region of immunoglobulines as do the 5 homologous SPA domains, but unlike the parental domain it does not bind to the Fab region. The term “affibody” is used to define a class of engineered proteins selected fro their specific binding activity towards a desired target and based on the Z domain.

See also “purification of antibodies” and “purification schemes”

See also Analogues/Derivatives/Mutagenesis/Variants     See also Resin regeneration

Staphylococcal protein A (SPA) is a protein that is found in nature anchored to the outer membrane of the gram-positive Staphylococcus aureus bacterium, the organisms which is commonly associated with medically significant human “Staph” infection. Functionally, SPA is well known for its ability to tightly, but reversibly bind to the constant region of IgG. This property has been widely used in the affinity purificaiton of antibodies. (Pyeser, US6,691,608).

Where Protein A Binds (epitopes): (see outline)

Recombinant production of Protein A

Recombinant production of Protein A in Gram positive bacterial strains is disclosed by Fahnestock in US4,617,266.  See also (Colbert, 5,151,350) for cloning of gene for protein A.

Jendeberg (J. Molecular Recognition, 8, 270-278 (1995) discloses that divalent (ZZ) forms of Protein A has a C terminal extension of 17 amino acids (VDANSRGSVDLQPSLSK).

Profy (US5,084,559) discloses modification of protein A gene to express a protein containing a single cysteine amino acid reisude at a defined position in the amino acid sequence. Protein A contains no cysteine residues in the WT.

Structure: see outline

Types of Media/Resins: See outline

Resin Regeneration See outline

Conditions/ Parameters: See outline

Applications

Streptavidin-Protein A chimeric Proteins: Sano (US 5328985) discloses a sterptavidin-Protein A chimeric protein in which the streptavidin binds biotin and the Protein A binds an antibody. Accordingly, one side specificity is caused by high affinity bind of the Protein A side to antibody molecules and the second side specificity is cuased by high affinity binding of the streptavidin side to any target biological material which contains biotin or which is capable of being biotinylated. The fusion protein can be used to purify target molecules onto solid supports. 

Biotin-Protein A chimeric Proteins: Patchornik (US20080108053) also discloses an antibody binding moiety such as protein A which is attached to at least one corrdinating moeity such as biotin. In order to initiate purification, the ligand attached to the coordinating moeity is added to the sample, allowing binding of the target molecule to the ligand and then a coordinator ion or molecule such as a metal is added to allow precipitation.