Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics

• Published in: Vox sanguinis Vol. 92; no. 4; pp. 338 - 350
• Main Authors: Laursen, I., Houen, G., Højrup, P., Brouwer, N., Krogsøe, L. B., Blou, L., Hansen, P. R.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.05.2007
• Subjects: affinity chromatography; Amino Acid Sequence; Animals; Antibodies; Chromatography, Affinity - methods; Cohn fraction III; Filtration - methods; Humans; Immunity, Innate; In Vitro Techniques; Mannose-Binding Lectin - blood; Mannose-Binding Lectin - isolation & purification; Mannose-Binding Lectin - therapeutic use; Mannose-Binding Protein-Associated Serine Proteases - genetics; Mannose-Binding Protein-Associated Serine Proteases - immunology; Molecular Sequence Data; nanofiltration; Nanotechnology; Plasma - chemistry; Plasma - immunology; plasma-derived MBL product; process; Rabbits; Safety; Superdex 200; Viruses - isolation & purification
• ISSN: 0042-9007; 1423-0410
• DOI: 10.1111/j.1423-0410.2007.00901.x

Background and Objectives  Mannan‐binding lectin (MBL) is an important component of the innate immune defence; it binds to carbohydrate structures on pathogenic micro‐organisms resulting in complement activation and opsonization. Individuals with low MBL levels are at risk of recurrent and severe infections. Substitution therapy with plasma‐derived MBL is a promising treatment of diseases associated with MBL deficiency. A first‐generation MBL product has been shown to be safe and well tolerated, and patients have benefited from MBL treatment. Following is a description of the development of a nanofiltered second‐generation MBL product from Cohn fraction III, with the use of a new affinity matrix for MBL purification and the characteristics of this improved product. Materials and Methods  Carbohydrate‐based gels were comparatively screened as affinity matrices. MBL was extracted from fraction III, and affinity purified on a Superdex 200 pg column. The eluted material underwent two virus reduction steps: filtration through Planova 20N and solvent/detergent treatment. It was further purified by anion‐exchange and gel‐filtration chromatography. The affinity eluate and the final MBL fraction were characterized by protein chemical, immunological, and functional assays. Results  In production scale, Superdex 200 pg was found to be superior to other carbohydrate‐based matrices, and MBL was affinity purified from fraction III with a yield of 70%. The viral safety was increased by performing a nanofiltration of the affinity eluate through Planova 20N with a minimal loss of MBL. The purity of the final MBL fraction was 53% excluding the MBL‐associated serine proteases (MASP). The product consisted of high‐oligomeric MBL, with two dominating forms, and with MASP‐1, ‐2, ‐3 and 19 kDa MBL‐associated protein (MAp19). Only a few protein impurities were present, the major being α2‐macroglobulin. MBL formed complexes with α2‐macroglobulin bridged by MASP‐1 covalently attached to the latter. The functional activity, assessed by mannan‐binding activity and opsonic function, was intact, whereas half of the C4 activating capacity was lost during the production process. Conclusion  A second‐generation MBL process was developed with an average yield of 50%. It was possible to nanofilter the MBL‐MASP complexes through Planova 20N with only a minor loss resulting in an increased safety profile of this MBL product.