Risk Management

Risk Management

Crude heparin, the starting material used in the manufacture of Celsus’ Heparin Sodium and the derivatives thereof, is extracted from the mucosal tissue of healthy hogs raised and slaughtered for food in compliant member nations of the Office International des Epizooties (OIE), The mission of OIE is to ensure transparancy of the world animal health situation based on the commitment of its 178 member countries and territories to promptly notify cases of animal disease detected in their territories. OIE is recognized as a reference agency of the World Trade Organization (WTO).

The extraction process involves protease hydrolysis to liberate the glycosaminoglycans (GAG) comprising heparin, chondroitin -, dermatan -, and heparan sulfate. The GAG in the form of a crude heparin is selectively adsorpted on an anion exchange resin, and recovered from a high salt eluate by solvent precipitation.

Figure 1: a “normal” prion molecule
Shown here, a “normal” prion molecule (PrPc). In abnormal prions (PrPsc), the “tail” portion of the protein folds in on itself. PrPsc is believed to hijack PrPc through protein-protein interactions to promote the further formation of PrPsc molecules.

Celsus certifies that effective May 15, 2012, all of its crude heparin starting material will have been druglisted and originates from FDA-registered drug establishments in countries that are recognized by the OIE as having either a negligible or a controlled risk of bovine spongiform encephalopaany thy (BSE), one of a group of diseases known as transmissible spongiform encephalopathy (TSE). Crude heparin, in the form of either crude heparin-on-resin, crude heparin eluate or crude heparin powder, is first quarantined at Celsus Laboratories, a USDA-inspected facility located in Sharonville, Ohio.

Before release to further processing, the crude heparin starting material: (a) must test negative for prion antigens (PrPsc/PrPc) by a validated, proprietary enzyme immunoassay (EIA) using a highly specific monoclonal antibody to an epitope among bovine, ovine and porcine prions; (b) is analyzed for animal species origin by a quantitative, proprietary polymerase chain reaction (PCR) procedure in which highly selective, species-specific DNA markers are used to authenticate porcine-origin and the appreciable absence of bovine adulteration; and (c) is tested for detectable oversulfated chondroitin sulfate and other extraneous polyanions by strong anion exchange high performance liquid chromatography (SAX-HPLC).

Figure 1 Porcine-Specific DNA Amplification from Porcine vs Bovine Crude Heparin by PCR

Amplification curves
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Celsus further certifies that Heparin Sodium is manufactured using a process validated for the removal or inactivation of TSE infectious agents, viral infectivity due to retroviruses, enterohepatic viruses and porcine parvoviruses; as well as pyrogen, bacteria, mould, yeast, extraneous salts and volatiles using manufacture and controls pursuant to Celsus drug master files reviewed by regulatory agencies in Australia, Canada, the European Union, Japan, and the United States.

To validate the process, model viruses (see Table 1 below) were chosen in compliance with EU regulatory guidelines prEN 12442-3, consistent with BS EN 22442-3, and the ICH Harmonized Tripartite Guidelines for Viral Safety Evaluation (ICH Q5A(R1)) taking into account process scale and controls, interference and cell toxicity.

Table 1 Physical, Chemical and Genomic Diversity of Viruses Studied
Table 1 Physical, Chemical and Genomic Diversity of Viruses Studied
Table 2: Properties of Model Viruses and Viral Assay
Table 2: Properties of Model Viruses and Viral Assay

In order to determine the robustness of the manufacturing process on viral elimination, model virus selection was based on the following criteria: (i) the potential pathogenic relevance to viruses in porcinederived heparin sodium; (ii) the genomic diversity of virus classifications; (iii) the physical-chemical diversity of virus structure and function properties and; (iv) the relative resistance of the viruses selected to physical-chemical inactivation procedures. In addition, the above selection process was based on literature review and discussions with viral expert consultants and officials at the U.K. Medicines Control Agency (MCA), renamed in 2003 the Medicines and Health care products Regulatory Agency (MHRA). Two steps of the Celsus heparin manufacturing process were evaluated for their virus reduction capabilities. In these studies, intermediate stage heparins were pulled from production, spiked with hightiter viruses and were then subjected to an Extended Exposure Treatment as per a USDA-approved protocol and a Limited Exposure Treatment to sanitize the product. To validate these two steps for virus elimination, the heparin was spiked with either HIV-1, poliovirus or porcine parvovirus. The method used to detect each virus, along with its limit of detection (LOD), can be seen in Table 2.

For the HIV studies, three different viral strains were used (RF, IIIB and the clinical isolate M301). Depending on the virus, titers ranged from about 1.5-5 x 106 infectious units per ml. HIV-1 was used to approximate a porcine endogenous retrovirus contaminant. For the polio studies (Mahoney strain), titers ranged from about 0.1 - 1.2 x 108/ml (average ~5 x 107/ml) and served as a model of enterohepatic viridae such as the family of Foot-and- Mouth associated viruses. For the Parvo virus studies, strain BL-006 (NADC-2), spiked at ~1 x 106, was used as a bone fide pathogenic porcine parvovirus. As summarized in Table 2 below, both steps of the manufacturing process eradicated all detectable traces of the virus input, i.e., no active virus or viral antigen was detectable. This is illustrated by the example of Figure 3 below taken from the poliovirus experiments using the Limited Exposure Treatment protocol .

Immunofluorescence imaging of Polio virus plaques
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Figure 3: Immunofluorescence imaging of Polio virus plaques.

Figure 3: Immunofluorescence imaging of Polio virus plaques. Panels A and B are low and high power fields, respectively, of cells infected by the heparin sample spiked with high-titer poliovirus that was not subjected to the Limited Exposure Treatment protocol. Abundant virus was detected in cells by immunofluorescence staining of viral antigens (bright yellow fluorescence). Panels C and D are the low and high power fields, respectively, of cells exposed to the heparin sample spiked with high titer poliovirus that was subjected to the Limited Exposure Treatment protocol. In contrast to the cell cultures of panels A and B, no virus growth occurred in the cells exposed to poliovirus-spiked heparin treated by the Celsus process.

For the TSE studies, samples were spiked at ~1 x 106/ml with TSE strain 263K and injected intracerebrally into living hamsters using serial dilutions down to 1 infectious unit/animal. The quantal assay used is sensitive to the presence of a single infectious unit which manifests as disease after ~ 250 days from initial exposure. All hamster groups receiving heparin spiked with TSE that was not treated by the Celsus process developed disease whereas animals receiving heparin spiked with TSE but treated by the Celsus process did not develop disease (see Table 2 below). Figure 4 shows the Western blot analysis of the brains of animals from the two groups analyzed for the presence of the infectious form of the prion antigen PrPsc. Hamsters that received heparin treated for TSE removal by the Celsus process (lanes 7-9) showed no evidence of TSE 263K prion antigen, i.e., they were free of infectious prions, while those receiving heparin not treated for TSE removal (lanes 1-6) exhibit high levels of brain-associated PrPsc. Lane 10, PrPsc antigen positive control.

Figure 4 Western Blot Analysis for TSE 263K PrPsc in Non-Infected vs. Infected Brain Tissues
Figure 4 Western Blot Analysis for TSE 263K PrPsc in Non-Infected vs. Infected Brain Tissues

Table 3 below summarizes the log reduction of infectious agents in Heparin Sodium by the Celsus manufacturing process. These data show that both the Limited and Extended Exposure Treatments used in the manufacture of Heparin Sodium yielded total eradication of all detectable infectious agents. The Total reflects the sum of the log reduction values (LRV) of the various treatments at two steps of a 37 step manufacturing process and therefore understates log reduction estimates by process.

Table 3: Log Reduction of Infectious Agents by Process
Table 3: Log Reduction of Infectious Agents by Process

Manufacture and Controls

Celsus has a policy to manufacture high quality derivatives of heparin through a rigorous commitment to operations in conformity with current good manufacturing practices (cGMP), an allowable alternative to ISO-certification under the EU Medical Device Directive.

Celsus Laboratories is FDA-registered to manufacture active pharmaceutical ingredients (API) in an establishment that has been approved by the USDA to handle restricted animal byproducts. Crude heparin, the starting material used in the manufacture of Celsus heparins, is extracted from the mucosal tissue of pigs. The extraction process involves protease hydrolysis to liberate the glycosaminoglycans (GAG) comprising heparin, chondroitin sulfates and heparan sulfate. The GAG in the form of crude heparin are selectively adsorbed on an anion exchange resin, and recovered from a concentrated salt eluate.

Crude heparin is processed by Celsus Laboratories pursuant to drug master files reviewed by regulatory agencies in Australia (DMF # 91/15229), Canada (DMF # 2003-009), the European Union (DMF # 17389-N1554), Japan (218MF10052), and the U.S. (DMF # 7944). Processing includes chemical treatments validated to eradicate TSE infectious agents, viral infectivity due to retroviruses, enterohepatic viruses and porcine parvovirus, bacterial endotoxin and other extraneous contaminants.

Final purification steps are performed in Hepa-filtered rooms monitored for microbial airborne particulates. The product is diluted in Purified Water USP and diafiltered against 1000 MWCO membranes to eliminate any excess salt and volatile matter, before final filtration using a 0.22 micron rated hydrophilic membrane filter. Bulk-lyophilization followed by size reduction yields a flakes with a porous and friable structure, which allows for easier reconstitution in water than crystallized or conventionally dried heparin. Finally, the dry API is tested by Quality Control for compliance with applicable compendial specifications and packaged for dispatch before one final audit of the entire process by Quality Assurance.

Quality Control and Quality Assurance

Compendial and other validated test procedures are performed at Celsus Laboratories to certify the strength, quality and purity of Heparin Sodium and various other derivatives of crude heparin. Celsus quality assurance personnel, who are authorized and directed to independently audit risk management, manufacture and controls, enforce conformity with cGMP.

Heparin Quality Control and Quality Assurance