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【血藍蛋白】生產疫苗、制備抗體-GMP/科研級別Keyhole Limpet Hemocyanin

KLH說明書//biosyncorp.com/klh/

 

傳統凍干粉狀態KLH會產生大量沉淀并可能導致在使用該產品過濾沉淀時損失高達30%-40%, 好的KLH制劑溶液應在結合前極少沉淀,從而可產生高效的偶聯和增強抗體反應。Biosyn公司于1995年創立于美國加州,是一家專業同時研發和生產高質量的科研和臨床級別血藍蛋白的生物醫藥公司。目前biosyn公司生產的血藍蛋白在大型國際醫藥公司中有約30%的市場占有率。Biosyn公司的KLH很好的解決了沉淀難溶的問題, 其KLH溶液產品的保質期可長達4年.


Biosyn公司簡介及產品介紹             

Biosyn公司于1995年創立于美國加州,是一家專業研發和生產高質量的科研級別臨床/GMP血藍蛋白的生物醫藥公司。目前biosyn公司生產的血藍蛋白在大型國際醫藥公司中有約30%的市場占有率。血藍蛋白來源于大鎖孔帽貝(透孔螺)的血淋巴,也被叫做匙孔血藍蛋白,是具有高度免疫原性的蛋白大分子,也是一種多功能蛋白,它不僅具有輸氧功能,而且還與能量的貯存、滲透壓的維持及蛻皮過程的調節有關,還具有酚氧化物酶活性和抗菌功能,被認為是節肢動物和軟體動物中的一種重要的免疫分子。


KLH用于疫苗的生產,免疫原的制備,KLH廣泛作為載體蛋白應用,交聯于半抗原和其他抗原,使它們具有更強的免疫原性以用于抗體的制備。由于其質量大,復雜性高,與其他載體蛋白相比KLH會引出更強的免疫應答。因為此蛋白來源于軟體動物,它在系統發生上遠離哺乳動物物種,在測定過程中產生會與典型靶點樣品發生交叉反應的抗體的可能性較小。作為大的蛋白質,KLH含有上百個伯胺和羧基基團可以成為與戊二醛,N-羥基琥珀酰亞胺酯和EDC等交聯試劑交聯的靶點。  

Product(產品) Grade(級別)  Uses(用途)  
Native KLH in Phosphate
buffer low endotoxin, tested
for biological safety, viral
clearance and metal ions
GMP Clinical grade For further processing in KLH-conjugate
vaccine manufacture for human and
animal use
Native KLH in Phosphate 
buffer, or other buffers low endotoxin
RG Routine immunological and antibody
studies and antibody production, initial
product development activity for
conjugate vaccine. Preparation of
Research Grade activated KLH
Native KLH in Phosphate
buffer or other buffers. No
endotoxin testing
RG Low cost research grade material for 
research uses
VACMUNE® liquid, 20 mg/mL
A novel formulation of immunocyanin
(KLH subunits) in water.
The immunocyanin concentration
is 20 mg/mL
HPG/CG • For further processing and in bulk
KLH-conjugate vaccine manufacture.
Antigens like carbohydrate, peptide,
protein, other molecules
• Conjugate vaccines for disease types
– cancer, AIDS, drug abuse,
hypotension, infectious disease and
others
• As lymphocyte helper antigen and/or
immunological tracer molecule, in
nonconjugate vaccine approaches,
(e.g. for dendritic cell-based bulk
vaccine manufacture)
• Manufacture of conjugate vaccines for
human and animal use
VACMUNE® liquid, 20 mg/mL
A novel formulation of immunocyanin
(KLH subunits) in water.
The immunocyanin concentration
is 20 mg/mL
RG • Routine immunological and antibody
studies and antibody production
• Initial product development activities
of vaccines
• Preparation of research grade
activated KLH subunits


 
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.

 

Product

Grade

Uses

Native KLH in Phosphate buffer low endotoxin, tested for biological safety, viral clearance and metal ions

GMP
Clinical
grade

For further processing in KLH-conjugate vaccine manufacture for human and animal use

Native KLH in Phosphate buffer, or other buffers low endotoxin

RG

Routine immunological and antibody studies and antibody production, initial product development activity for conjugate vaccine. Preparation of Research Grade activated KLH

Native KLH in Phosphate buffer or other buffers. No endotoxin testing

RG

Low cost research grade material for research uses

RG=Research Grade
Biosyn公司網址: //biosyncorp.com/klh/
中國獨家授權代理:
深圳市華拓生物科技有限公司(
京东彩票合买保底 www.kcqnjv.com.cn
 

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