resumo
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 (alpha-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.
palavras-chave
AQUEOUS 2-PHASE SYSTEMS; MONOCLONAL-ANTIBODIES; PROTEIN SEPARATION; FRACTIONATION; ALBUMIN; SODIUM
categoria
Engineering
autores
Almeida, MR; Ferreira, F; Domingues, P; Coutinho, JAP; Freire, MG
nossos autores
Ana Mafalda Rodrigues Almeida Rocha
Investigador
Mara Guadalupe Freire Martins
Investigador CoordenadorGrupos
G4 - Materiais Renováveis e Economia Circular
G5 - Materiais Biomiméticos, Biológicos e Vivos
Projectos
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
CICECO - Aveiro Institute of Materials (UIDP/50011/2020)
Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)
Towards the use of IgY antibodies as alternative therapeutics (PureIgY)
agradecimentos
This work was developed within the scope of the project CICECOAveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 and LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC). This project has also received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 899921).