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The didecamer consists of 20 subunits and occurs in two different forms named KLH1 and KLH2. The subunits are biochemically 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. There are eight functional units in the KLH subunits, KLH1 and KLH2. 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 binds molecular oxygen. The copper is in the cuprous [Cu(I)] state, and oxygen binding generates light absorbance in the near ultraviolet around 340 nm and imparts the characteristic blue color to the molecule. The deoxygenated molecule is colorless.

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 come off readily. Further differences between the two hemocyanin subunits are revealed by hydrogen peroxide treatments.

 

Native KLH

The high molecular weight, native KLH, has been extensively over the last several years, and is still being used. Native KLH has been manufactured and supplied by many chemical suppliers.

However, most such KLH 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 dead animals shipped frozen. The product characteristics are also not very good and could lead to precipitation during reconstitution of lyophilized KLH or short shelf-life of one year or less for liquid preparation. 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.

To achieve the desired safety, quality and consistency of native KLH, we use 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 have been developed. In addition to this, we established aquaculture developmental activities to spawn, culture and grow the animals. The hemocyanin production process is thus environmentally friendly. Moreover, the non-lethal technology allows for the development and institution of appropriate mariculture/aquaculture techniques to ensure future stable source of the raw material.

The end result of these technology developments in the animal handling, bleeding and manufacturing process was the production of low endotoxin and high concentration native KLH of consistent high quality in a phosphate buffer containing no extraneous metal ions for product stabilization, however with improved stability.

The native KLH is supplied as High Purity Grade and Research Grade. The GMP grade material is ideally suited for use as a carrier protein in the manufacture of vaccines for human use. 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 gentaur are listed in the table above.

 

General Conjugation Procedures
- Using Hemocyanin and Immunocyanin Products

Why Conjugate?

Substances of low molecular weight (haptens) and some high molecular weight proteins alone will not generate antibodies or are not highly immunogenic when injected into an animal. Therefore, the haptens are usually conjugated to a large protein molecule known as a "carrier."

The carrier facilitates the production of antibodies in the following ways:

  • The increased size of the hapten-carrier complex is sufficiently large to be recognized and engulfed by the antigen presenting cells of the immune system.

  • The protein carrier molecules contain sequences that are T cell helper epitopes that stimulate the proliferation of helper T cells. Interaction of helper T cells with B cells is essential for a strong immune response.

  • Since carrier proteins are foreign to the animal, they enhance the response from the immune system.

Choosing a Carrier Protein

The choice of the carrier protein depends on the intended use of the hapten-carrier protein complex. For routine animal studies for research investigation and possibly development of immunological assays, there are a number of proteins that have been used as carriers, including Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), ovalbumin, and Tetanus toxoid. KLH and BSA are probably the most commonly used.

However, for human clinical studies the most widely used carrier proteins are Keyhole Limpet Hemocyanin, Tetanus toxoid or Diphtheria toxoid. The critical parameters in the selection of the protein are elucidation of the necessary immune response for the intended clinical use and the toxicity of the carrier protein and the conjugate, i.e. minimal secondary complications. These are generally evaluated during the early research and pre-clinical studies. KLH has been the most widely used protein due to the consistent yield of enhanced immune response with a wide variety of haptens and also protein molecules.

KLH is one of the largest foreign proteins that is most different from the human and generates the best immune response. When immunized with a hapten-KLH conjugate, antibodies are also generated to the KLH. Therefore, it is necessary to use a hapten-conjugate that is different from the KLH used for your assays. For example, a KLH conjugate should be used for immunizations and a BSA conjugate for assays. More recent investigations suggest that biosyn hemocyanin from abalone and horse shoe crab may be used as an additional carrier protein.

GMP KLH has many advantages over other commercial grade material that can be procured from other vendors. The major distinction lies in:

  • Environmentally friendly process used in the extraction of hemocyanin from animals.

  • 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 most commercial KLH suppliers.

  • GMP hemocynin and final products are therefore of high purity and low endotoxin quality.

  • GMP KLH subunits, Immunocyanin, are of low molecular weight (400 kDa) and are a completely characterized product.

  • GMP 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.

  • GMP products are extensive tested for safety and lack of viruses including hepatitis A, B, C, vibrio species, rota virus and Norwalk viruses.

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