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

Abstract

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 alpha2-macroglobulin. MBL formed complexes with alpha2-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.

• Authors: I Laursen, G Houen, P Højrup, N Brouwer, L B Krogsøe, L Blou, P R Hansen
• Journal: Vox sanguinis (Vox Sang) Vol. 92 Issue 4 Pg. 338-50 (May 2007) ISSN: 0042-9007 [Print] England
• PMID: 17456158 (Publication Type: Journal Article)

Chemical References

• Antibodies
• Mannose-Binding Lectin
• MASP1 protein, human
• MASP2 protein, human
• Mannose-Binding Protein-Associated Serine Proteases

Topics

• Amino Acid Sequence
• Animals
• Antibodies
• Chromatography, Affinity (methods)
• Filtration (methods)
• Humans
• Immunity, Innate
• In Vitro Techniques
• Mannose-Binding Lectin (blood, isolation & purification, therapeutic use)
• Mannose-Binding Protein-Associated Serine Proteases (genetics, immunology)
• Molecular Sequence Data
• Nanotechnology
• Plasma (chemistry, immunology)
• Rabbits
• Safety
• Viruses (isolation & purification)