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