Membrane affinity chromatography for analysis and purification of biopolymers

Abstract

KeyWordsColumn liquid chromatography Membrane stationary phase Immunoaffinity analysis Affinity efficiency Adsorption capacity SummaryAffinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only about 2.5 min for analysis at a flow-rate of 1.0 mL min -1. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 1 mL rain-within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid. It can also be used for micro-scale purification ofbiopolymers.

Alkaline treatment of the cellulose fiber affecting membrane column behaviour for high-performance immunoaffinity chromatography

“…It can be estimated that the fraction of the total accessible volume (F e ) on those two columns is 24% and 40%, respectively, which is relatively lower than the 60% measured on the column with the Superose medium. The above result is also supported by the fact that the adsorbed capacity of the proteins on the membrane based upon the immobilized metal affinity and the protein A immunoaffinity columns was much lower than that on the Superose based columns (Kasai et al, 1986;Zhou et al, 1999b). However, the alkali treatment of cellulose composite membrane greatly increases the accessible volume to proteins (Gao and Tang, 1996).…”

“…The immunoaffinity analysis was performed by directly injecting human and dog plasma diluted 10 times with loading buffer, which indicates that the membrane media have very good mass flux for the compounds with different molecular weight up to 900 kDa for IgM in plasma. In fact, human IgG and human IgM were successfully immobilized on those media for immunoaffinity analysis of goat polyclonal antibodies to human IgG and human IgM (Zhou et al, 1999b). By comparison of the peak area of human IgG in Figs 5(a) and 6(a) with that of the retained peak in Figs 5(b) and 6(b), the amount of human IgG in human serum and dog IgG in dog serum both can be determined using the pure human IgG as the standard sample when they are assumed to have the same absorptivity at 280 nm.…”

Protein A immobilized monolithic capillary column for affinity chromatography

“…Hexanediamine is a popular spacer arm and the adsorption capacity of the column is greatly increased if the spacer arm is coupled to the matrix. However, hydrophobic interaction will occur between the spacer arm and the protein to be separated, which often result in the non-specific adsorption of separated proteins [30,31]. In this work, as shown in Fig.…”

Affinity membrane chromatography for the analysis and purification of proteins

“…[ ] proteins 59,60,143,144,164 With the development of biotechnology in recent years, fast analysis and purification of proteins is becoming more and more indispensable for the optimization and control of biological process. Affinity membrane columns compatible with HPLC instruments were prepared in our laboratory by immobilization of various ligands of Protein A, human IgG, human IgM, DEAE, IDA-Cu 2q , and pectinase on celluloserGMA composite membrane.…”