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

I Laursen, G Houen, P Højrup, N Brouwer, L B Krogsøe, L Blou, P R Hansen

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 2007-May
OriginalsprogEngelsk
TidsskriftVox Sanguinis
Vol/bind92
Udgave nummer4
Sider (fra-til)338-350
Antal sider12
ISSN0042-9007
DOI
StatusUdgivet - 1. maj 2007

Fingeraftryk

Mannose-Binding Protein-Associated Serine Proteases
Mannans
Safety
Proteins
Gel Chromatography
Viruses

Citer dette

Laursen, I ; Houen, G ; Højrup, P ; Brouwer, N ; Krogsøe, L B ; Blou, L ; Hansen, P R. / Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics. I: Vox Sanguinis. 2007 ; Bind 92, Nr. 4. s. 338-350.
@article{01bcd020d94e11dc860c000ea68e967b,
title = "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.",
keywords = "Amino Acid Sequence, Animals, Antibodies, Chromatography, Affinity, Filtration, Humans, Immunity, Natural, Mannose-Binding Lectin, Mannose-Binding Protein-Associated Serine Proteases, Molecular Sequence Data, Nanotechnology, Plasma, Rabbits, Safety, Viruses",
author = "I Laursen and G Houen and P H{\o}jrup and N Brouwer and Krogs{\o}e, {L B} and L Blou and Hansen, {P R}",
year = "2007",
month = "5",
day = "1",
doi = "10.1111/j.1423-0410.2007.00901.x",
language = "English",
volume = "92",
pages = "338--350",
journal = "Vox Sanguinis",
issn = "0042-9007",
publisher = "Wiley-Blackwell",
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Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics. / Laursen, I; Houen, G; Højrup, P; Brouwer, N; Krogsøe, L B; Blou, L; Hansen, P R.

I: Vox Sanguinis, Bind 92, Nr. 4, 01.05.2007, s. 338-350.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics

AU - Laursen, I

AU - Houen, G

AU - Højrup, P

AU - Brouwer, N

AU - Krogsøe, L B

AU - Blou, L

AU - Hansen, P R

PY - 2007/5/1

Y1 - 2007/5/1

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Animals

KW - Antibodies

KW - Chromatography, Affinity

KW - Filtration

KW - Humans

KW - Immunity, Natural

KW - Mannose-Binding Lectin

KW - Mannose-Binding Protein-Associated Serine Proteases

KW - Molecular Sequence Data

KW - Nanotechnology

KW - Plasma

KW - Rabbits

KW - Safety

KW - Viruses

U2 - 10.1111/j.1423-0410.2007.00901.x

DO - 10.1111/j.1423-0410.2007.00901.x

M3 - Journal article

C2 - 17456158

VL - 92

SP - 338

EP - 350

JO - Vox Sanguinis

JF - Vox Sanguinis

SN - 0042-9007

IS - 4

ER -