Session: Parallel session 7 - Club jeune SFSM

Comparative Analysis of Laboratory-Scale Immunoglobulin G Purification Methods from Human Serum

Louisa BOUREL¹, Vincent SOBANSKI^1,4,6, Fabrice BRAY², Solange VIVIER¹, Stéphanie FLAMENT², Lucile GUILBERT^1,3, Aurélien CHEPY^1,4, Christian ROLANDO^2,5, David LAUNAY^1,4, Sylvain DUBUCQUOI^1,3

¹Univ.Lille, Inserm, CHU Lille, U1286-INFINITE—Institute for Translational Research in Inflammation, 59000 Lille, France, Lille, France
²Univ. Lille, CNRS, UAR 3290 -MSAP -Miniaturisation pour la Synthèse, l'Analyse et la Protéomique, F-59000 Lille, France, Lille, France
³CHU Lille, Institut d’Immunologie, Centre de Biologie Pathologie, 59000 Lille, France, Lille, France
⁴CHU Lille, Département de Médecine Interne Et Immunologie Clinique, Centre de référence des Maladies Auto-Immunes et Auto-inflammatoires Systémiques rares de l'Adulte du Nord, Nord-Ouest, Méditerranée et Guadeloupe (CeRAINOM), 59000 Lille, France, Lille, France
⁵Shrieking Sixties, 1-3 Allée Lavoisier, F-59650 Villeneuve-d'Ascq, France, Villeneuve-d'Ascq, France
⁶Institut Universitaire de France (IUF), 75005 Paris, France, Paris, France

Introduction: Immunoglobulin G (IgG) purification is essential for evaluating its role in autoimmune diseases, which are defined by the presence of autoantibodies. Affinity chromatography with protein G is widely considered the optimal technique for laboratory-scale purification. However, this technique has some limitations, including the exposure of IgG to low pH, which can compromise the quality of purified IgG.

Objective: This study aimed to evaluate different methods for isolating IgG from serum, focusing on yield, purity, and quality of the purified IgG.

Methodology: IgG from sera was purified using the following techniques: protein G affinity chromatography, Melon^TM Gel, caprylic acid-ammonium sulfate (CA-AS) precipitation, anion exchange chromatography with diethylaminoethyl (DEAE) following AS precipitation, and AS precipitation alone. Purification yields were determined by turbidimetry. Purity was assessed using 1D-SDS-PAGE-LC-MS/MS on a Q Exactive Plus Orbitrap, with data analysis performed using MaxQuant and Perseus. The avidity of purified IgG for two selected targets (SARS-CoV-2 and topoisomerase-I) was determined using a modified ELISA. Preservation of IgG glycosylation after purification was evaluated by LC-MS/MS, with glycopeptides identified using Byonic and quantified using pGlycoQuant.

Results: The alternative purification techniques yielded higher IgG amounts compared to protein G, but disparities in IgG purity were observed. Despite these differences, the quality of IgG remained consistent, as evidenced by the similarities in IgG avidity for selected targets before and after purification across all methods, as well as the preservation of IgG glycosylation.

Conclusion: Our work provides valuable insights for future studies on IgG function by recommending alternative purification methods that offer advantages in terms of yield, time efficiency, cost-effectiveness, and milder pH conditions than protein G.