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KLH: Keyhole Limpet Hemocyanin
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.
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.
Keyhole Limpet Hemocyanin Structure
Prof. Dr. Jürgen Markl, Direktor Zoologisches Institut der Johannes Gutenberg-Universit?t 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.
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.
|Native KLH in Phosphate buffer low endotoxin, tested for biological safety, viral clearance and metal ions||
|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
Why use biosyn KLH?
biosyn KLH has many advantages over other commercial grade material that can be procured from other vendors. The major distinction lies in biosyn's:
Environmentally friendly process used in the extraction of hemocyanin from animals.
biosyn returns the animals back to its natural habitat, as it is a humane approach as well ensures that the animals recovers readily and more impotantly assures the product quality and reproducibility leading to no potential undefined, difficult to characterize, structural alterations to the hemocyanin molecule.
biosyn uses a proprietary methodology and has built-in a test procedure for harvesting and handling of live animals, unlike the use of dead or frozen animals or lethal extraction procedure used by some commercial KLH suppliers.
biosyn hemocynin and final products are therefore of high purity and low endotoxin quality.
biosyn KLH subunits, immunocyanin, are of low molecular weight (400 kDa) and are a completely characterized product.
biosyn manufactures and packages KLH products to meet specific customer requirements including userfriendly, ready-to-use preparation and packaging.
biosyn hemocyanin and immunocyanin are formulated in either pure buffer systems like phosphate buffer or in water and packaged as small aliquots or bulk quantities. This offers customers major advantages, i.e., flexibility of use, choice of buffer in early developmental studies, and avoidance of issues associated with reconstitution of dry powder product obtained from other vendors.
biosyn Immunocyanin in water is a unique formulation and overcomes one of the major drawbacks of using KLH in clinical and commercial grade conjugate vaccine products.
biosyn clinical grade products are extensive tested for safety and lack of viruses including hepatitis A, B, C, vibrio species, rota virus and Norwalk viruses.