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

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.

Minimally processed fresh frozen human reference sera: preparation, testing, and application to international external quality assurance.

The preparation of unmodified or minimally processed fresh frozen human sera is described, as well as the previous use of such sera, e.g. in Nordic and international external quality assurance (EQA) activities. The unmodified serum is prepared from fresh donors' blood collected in dry bags and allowed to coagulate. The serum is collected "on the clot", pooled, filtered, mixed, dispensed in polypropylene vials and frozen at -80 degrees C without further processing. Some batches were slightly modified by spiking or dilution. Critical steps of the production and use of the sera are described and improvements are discussed. A total of 34 different batches have been prepared since 1985. Results from homogeneity and stability studies are presented. The studies cover 18 routine components in serum stored at +4 degrees C to 37 degrees C for up to 34 days. Good stability was observed for storage of all components, with the exception of triglyceride. Amylase, creatininium, glucose, gamma-glutamyltransferase, urate (and perhaps carbamide) showed deterioration after 13 days of incubation at 37 degrees C. The long-term stability at -80 degrees C is reviewed and new data are presented, e.g. as consensus values from EQA schemes, where the same serum has been sent out three times over 5 years, and from reference measurement procedure values that have been assigned twice with an interval of 5 years. Furthermore, a 10-year stability study has been started.

Human plasma-derived mannose-binding lectin: a phase I safety and pharmacokinetic study.

Mannose-binding lectin (MBL) is an important component of innate immunity that can bind to certain sugar residues on the surface of many types of pathogenic micro-organisms. On binding, MBL generates opsonic activity mainly through activation of the complement system. Genetically determined MBL deficiency is very common and can be associated with increased susceptibility to a variety of infections, especially in children and immunosuppressed individuals. The potential benefits of MBL reconstitution therapy therefore need to be evaluated. We have carried out a phase I safety and pharmacokinetic study on 20 MBL-deficient healthy adult volunteers. The MBL was prepared from plasma of nonremunerated, voluntary Danish donors tested and found negative for hepatitis B surface antigen, antibodies to human immunodeficiency virus (HIV) and hepatitis C virus. Each volunteer received a total of 18 mg of MBL in three 6 mg doses given intravenously, once weekly over a period of 3 weeks. The volunteers were closely monitored at the University Hospital in Reykjavik for 8 h after each infusion and daily thereafter for 5 days after each infusion. No adverse clinical or laboratory changes were observed in any of the 20 participants, and frequent measurements did not reveal any signs of infusion-associated complement activation. No antibodies to MBL, HIV or hepatitis viruses were observed 24 weeks after the last infusion. Serum MBL levels increased up to normal levels (1200-4500 ng/ml) immediately after each infusion, but the half-life of the infused MBL was highly variable, ranging from 18 to 115 h (mean 69.6). It is concluded that infusion of purified MBL as prepared by Statens Serum Institut (SSI) is safe. However, adults have to be given at least 6 mg twice or thrice weekly for maintaining protective MBL levels assumed to be about 1000 ng/ml.