血蓝蛋白丨美国biosyn公司

Biosyn公司于1995年创立于美国加州，是一家专业研发和生产高质量的科研级别和临床/GMP血蓝蛋白的生物医药公司。目前biosyn公司生产的血蓝蛋白在大型国际医药公司中有约30%的市场占有率。血蓝蛋白来源于大锁孔帽贝（透孔螺）的血淋巴，也被叫做匙孔血蓝蛋白，是具有高度免疫原性的蛋白大分子，也是一种多功能蛋白，它不仅具有输氧功能，而且还与能量的贮存、渗透压的维持及蜕皮过程的调节有关，还具有酚氧化物酶活性和抗菌功能，被认为是节肢动物和软体动物中的一种重要的免疫分子。

KLH用于疫苗的生产，免疫原的制备。KLH广泛作为载体蛋白应用，交联于半抗原和其他抗原，使它们具有更强的免疫原性以用于抗体的制备。由于其质量大，复杂性高，与其他载体蛋白相比KLH会引出更强的免疫应答。因为此蛋白来源于软体动物，它在系统发生上远离哺乳动物物种，在测定过程中产生会与典型靶点样品发生交叉反应的抗体的可能性较小。作为大的蛋白质，KLH含有上百个伯胺和羧基基团可以成为与N-羟基琥珀酰亚胺酯和EDC等交联试剂交联的靶点。

biosyn Native KLH

* HPG = High Purity Grade; RG = Research Grade

biosyn Immunocyanin Products

* HPG = High Purity Grade; RG = Research Grade; CG = Clinical Grade

1、KLH
biosyn hemocyanin products
biosyn is a world leader in the manufacture of high quality, clinical/GMP and research grade hemocyanin products derived from the hemolymph of the sea snail, Meghathura crenulata, also commonly known as the Giant Keyhole Limpet.

The hemocyanin derived from this snail is known Keyhole Limpet Hemocyanin (KLH).
.biosyn hemocyanin - first hemocyanin product for the treatment of bladder cancer
.biosyn hemocyanin and immunocyanin, KLH subunits, as a preferred carrier protein of the conjugate vaccine
.immunocyanin - high quality - GMP/Clinical grade - Drug Master File
.biosyn hemocyanin products are eco-friendly
.Recombinant technology development

 

2、KLH/KLH: Keyhole Limpet Hemocyanin

KLH: Keyhole Limpet Hemocyanin - biosyn native KLH
The hemocyanin produced from the hemolymph sera of the marine mollusk Giant Keyhole Limpet, Megathura crenulata, has been in use and marketed as a crude or partially purified product for over 40 years by some chemical companies. This molecule is well recognized and commonly abbreviated as KLH. The crude research grade KLH is used in antibody production in animals against antigens. The animal sera containing the antibody for the antigen are further processed and used as immunological reagents or in immunological assays. The partially purified product has also been used in early human trials for immune status evaluation.

KLH structure
The native KLH is a cylindrical copper containing blue protein with a molecular mass ranging from 8-32 MDa (Million Dalton). Electron microscopy structural studies reveal presence of two oligomeric forms:

.Didecamers with a molecular mass of 8 MDa, are present in the form of hollow cylinders. They are approximately 35 nm in diameter and 40 nm in length.

.Multidecamers with a molecular mass of 12-32 MDa, present as stacked decamers (multidecamers) of varying length, added on one or both sides of a "nucleating" didecamer. Single decamers exhibit rectangular side views.

Prof. Dr. Jürgen Markl, Direktor Zoologisches Institut der Johannes Gutenberg-Universität Mainz, Germany

The didecamer consists of 20 subunits and occurs in two different Isoforms named KLH1 and KLH2. The subunits are biochemically and immunologically distinct. Analysis of KLH by native-PAGE gives two characteristic bands, one corresponding to KLH1 with an apparent molecular mass of about 390 kDa (kilo Dalton) and the other corresponding to KLH2 with an apparent molecular mass of 350 kDa.

Each subunit of both Isoforms, KLH1 and KLH2, consists of eight paralogous functional units (FU), named 'a' to 'h', whereby the FUs a-f form the wall of the cylinder and g-h build up the collar-complex of the molecule. The molecular mass of the functional units is about 50 kDa. Based on this information it is expected that the molecular masses of the KLH1 and KLH2 subunits are approximately 400 kDa. The functional unit (FU) within the subunit contains a binuclear copper binding site that reversibly binds molecular oxygen.

The copper is in the cuprous [Cu(I)] state, and oxygen-binding generates light absorbance in the near ultraviolet around 343 nm and imparts the characteristic blue color to the molecule. The deoxygenated molecule is colorless.

Peptide sequence
The peptide sequence surrounding the two copper-binding sites is highly conserved, with three copper-liganding histidines in both cases. The active site geometry and molecular architecture of the mollusk hemocyanin differ from those of arthropod hemocyanin.
Removal of second copper from mollusk hemocyanin by cyanide ions is both slower and more difficult compared to arthropod hemocyanin, where all the copper comes off readily. Further differences between the two hemocyanin subunits are revealed by hydrogen peroxide treatments.

3、KLH / biosyn native
KLH biosyn native KLH, low enodoxin content
The high molecular weight, native KLH, is extensively used and has been used over the last several years. Native KLH has been manufactured and supplied by some chemical suppliers.

However, most available KLH formulations are not of the required quality and consistency suitable for use in human clinical trials. Typically, the endotoxin content in such products is very high as the hemolymph sera production is done by cutting open the animals or extraction from frozen animals. The product characteristics are also not very good and could lead to precipitation during reconstitution of lyophilized KLH. It is well documented that either freezing or lyophilization of native KLH leads to loss in activity.

Characterization of native KLH is also an issue as the large molecular weight of 8-32 MDa does not allow use of standard biochemical methods for routine quality control, which is further complicated by the use of excessive metal ions like calcium and magnesium, presumably to stabilize the molecule.

Right from the start of the hemocyanin development work in 1985, biosyn had identified these to be critical issues and developed appropriate strategies to overcome these limitations. To achieve the desired safety, quality and consistency of native KLH, biosyn has developed the non-lethal hemolymph sera collection procedure from live animals and instituted necessary animal handling and quarantine techniques. Quality control methodologies for routine production of this critical raw material has been developed.

The end result of these technology developments in the animal handling, bleeding and manufacturing process was the production of biosyn low endotoxin and high concentration native KLH of consistent high quality in a phosphate buffer.

The biosyn native KLH is supplied as High Purity Grade and Research Grade (no endotoxin testing). The GMP grade material is ideally suited for use as a carrier protein in the manufacture of vaccines for human use. It is tested for biological safety, viral clearance and heavy metal ions.

The Research Grade material is suited for use in vaccine product development activities and also for routine immunological studies, antibody production, production of activated KLH and other developmental activities. The native KLH formulations currently available from biosyn are listed in the table.

 

RG=Research Grade
中国独家授权代理：
深圳市华拓生物科技有限公司（www.otwobiotech.com）
相关产品：血蓝蛋白抗体