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|Title:||Towards the purification of IgY from egg yolk by centrifugal partition chromatography|
|Author:||Almeida, Mafalda R.|
Domingues, Pedro dimas
Coutinho, João A. P.
Freire, Mara G.
Aqueous biphasic systems
Centrifugal partition chromatography
|Abstract:||Although their high potential as alternative biopharmaceuticals, less than 2% of the total polyclonal antibodies produced worldwide correspond to immunoglobulin Y (IgY) due to the difficulties in isolating them from egg yolk (complex biological matrix). In this work, the water-soluble proteins fraction (WSPF) of egg yolk was first obtained and the proteins present identified by one-dimensional gel electrophoresis (SDS-PAGE) and label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). The egg yolk WSPF was then applied to create aqueous biphasic systems (ABS) composed of polyethylene glycol 1000 g·mol−1 (PEG 1000) and K2HPO4/ KH2PO4 buffer, followed by centrifugal partition chromatography (CPC) to purify IgY. The characterization of the WSPF showed the presence of six major proteins: the target antibody IgY, serum albumin (α-livetin), ovalbumin, ovotransferrin, vitellogenin 1 and vitellogenin 2. The results obtained by ABS revealed a high affinity of all proteins to the polymer-rich phase. However, by changing the PEG and salt concentrations, a higher selectivity was observed for IgY, with the remaining proteins partitioning between the two phases. The best ABS were applied in CPC, finally allowing a multi-stage partition and to the technology scale-up. The CPC operating conditions were optimized, allowing to obtain IgY with 50.6% of purity.|
|Appears in Collections:||CICECO - Artigos|
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