Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

A new process of IgG purification by negative chromatography: Adsorption aspects of human serum proteins onto ω-aminodecyl-agarose

“…Since HSA (the most abundant protein in human serum) was adsorbed onto adsorbents containing amine groups (TREN-and -aminohexyl-agarose [20,21]), it was expected that aminodecyl immobilized in agarose could also provide adsorption of HSA. In order to select a buffer condition that would favor the adsorption of HSA from human serum, chromatographic experiments were carried out by loading 0.3 mL of human serum diluted 20 times with three loading buffers at different pH values (Mops, Mes, and Hepes) onto a 3.0 mL bed of -aminodecyl-agarose.…”

“…As an example of mixed-mode chromatography, histidine-grafted aminohexyl-Sepharose was used for separation of HSA and its nonenzymatically glycated isoforms [19]. As other examples, the polyamines Tris(2-aminoethyl)amine (TREN) and -aminohexyl-grafted agarose and histidine-grafted aminohexyl-Sepharose were used as negative affinity adsorbents for purification of IgG from human serum and plasma [19][20][21][22]. Negative chromatography aims to obtain the product in the flowthrough and washing steps, while the contaminants or impurities remain adsorbed and can be recovered in elution steps [23].…”

“…Negative chromatography aims to obtain the product in the flowthrough and washing steps, while the contaminants or impurities remain adsorbed and can be recovered in elution steps [23]. Using TREN-agarose and -aminohexyl-agarose as negative adsorbents, our research group obtained high purity IgG in nonretained fractions (flowthrough and washing) with recoveries of around 75% [20,21].…”

“…The interactions between polyamine ligands and biomolecules are probably a mixed mode of an electrostatic and hydrophobic nature [27]. Depending on the length of the polyamine carbonic chain, hydrophobic interactions may also occur and can be exploited in the adsorption process for biomolecule purification [21]. A proper combination of interactions of different natures can result in the achievement of high selectivity, reducing the number of downstream processing steps and, in some cases, solving intractable purification problems [18,28].…”

CROMATOGRAFIA NEGATIVA EM AMINOHEXIL- AGAROSE: SEPARAÇÃO DE FRAGMENTOS Fab HUMANO

“…No entanto, dependendo do tamanho de sua cadeia carbônica e do braço espaçador utilizado, as interações hidrofóbicas entre a molécula alvo e o ligante não podem ser excluídas (Houen et al, 2001;Souza et al, 2010).…”