Towards the purification of IgY from egg yolk by centrifugal partition chromatography

• Published in: Separation and purification technology Vol. 299; p. 121697
• Main Authors: Almeida, Mafalda R., Ferreira, Filipe, Domingues, Pedro, A. P. Coutinho, João, Freire, Mara G.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.10.2022
• Subjects: Aqueous biphasic systems; Centrifugal partition chromatography; Immunoglobulin Y; Purification; Aqueous biphasic systems; Purification; Centrifugal partition chromatography; Immunoglobulin Y
• ISSN: 1383-5866; 1873-3794
• DOI: 10.1016/j.seppur.2022.121697

•The water-soluble protein fraction (WSPF) of egg yolk has six major proteins.•Aqueous biphasic systems are used to purify immunoglobulin Y (IgY) from the WSPF from egg yolk.•The effect of the pH and content of phase-forming components was evaluated.•A high affinity of IgY to the polymer-rich phase was observed, with the remaining proteins partitioning between the two phases.•The most promising ABS were applied in centrifugal partition chromatography to purify IgY. 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.