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


UZUN L. , TÜRKMEN D. , KARAKOC V., Yavuz H. , DENİZLİ A.

JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION, cilt.22, sa.17, ss.2325-2341, 2011 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 22 Konu: 17
  • Basım Tarihi: 2011
  • Doi Numarası: 10.1163/092050610x538731
  • Dergi Adı: JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION
  • Sayfa Sayıları: ss.2325-2341

Özet

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 mu m. 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. (C) Koninklijke Brill NV, Leiden, 2011