Performance of Protein-A-Based Affinity Membranes for Antibody Purification

Abstract

The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5-10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL-protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL-protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL-protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption-elution cycles.

Intensified Downstream Processing of Monoclonal Antibodies Using Membrane Technology

“…[ 56 ] Boi (2008) used a novel protein A membrane chromatography device based on regenerated cellulose support to capture mAb and achieved a maximum DBC of 9 mg mL −1 , which was at least 4 times lower than commercially available protein A resins. [ 57 ] With the benefit of material science technologies, novel membrane supports such as protein‐A‐attached poly(hydroxyethyl methacrylate‐ N ‐methacryloyl‐ l ‐alanine) membranes [ 58 ] and lectin modified poly(2‐hydroxyethyl methacrylate‐ethylene dimethacrylate) hydrogel membranes [ 59 ] show potential as capture devices. The commercial applicability of these membrane supports depends on factors such as their improved pore structure, low non‐specific interactions, chemical stability, re‐usability, proven performance under representative process conditions, and most importantly, their cost.…”

Poly(hydroxyethyl methacrylate) based affinity membranes for in vitro removal of anti-dsDNA antibodies from SLE plasma

“…Preparation of PHEMAAL membranes had been reported in our previous report [21]. The PHEMAAL membrane was prepared as described in the above section.…”

Bio-functionalized nanofibrous membranes as a hybrid platform for selective antibody recognition and capturing

“…[1][2][3] As a result, versatile technologies like precipitation, ultraltration and bio-chromatography have been exploited for antibody purication and polishing in order to facilitate the downstream processes. [5][6][7] The protein A, a single polypeptide chain, consists of four high-affinity binding sites capable of specically reacting with the Fc region from IgG. In general, the affinity interactions include ion exchange (IEX), hydrophobic interaction (HIC), immobilized metal affinity chromatography (IMAC), and bio-recognition with protein ligands, among which protein ligand (such as protein A, G, or A/G) based affinity chromatography exhibits the best purication performance in terms of the highest selectivity and binding capacity toward immunoglobulin G (IgG).…”

“…In general, the affinity interactions include ion exchange (IEX), hydrophobic interaction (HIC), immobilized metal affinity chromatography (IMAC), and bio-recognition with protein ligands, among which protein ligand (such as protein A, G, or A/G) based affinity chromatography exhibits the best purication performance in terms of the highest selectivity and binding capacity toward immunoglobulin G (IgG). [5][6][7] The protein A, a single polypeptide chain, consists of four high-affinity binding sites capable of specically reacting with the Fc region from IgG. Similar to the protein A from cell wall proteins, the protein G contains two IgG-binding domains as well as albumin binding sites.…”