Status: approved; marketed

Organizations involved:

Protherics PLC – Manuf.; R&D; Tech.

BTG plc – Parent; World mark.

Altana Inc. – Former

Nycomed Group – Former

Byk Gulden S.A. – Parent

Mayne Pharma Ltd. – Manuf. other

Hospira – Parent

Chesapeake Biological Labs/ (CBL) – Manuf. other

Cangene Corp. – Parent

Cross ref: See the entries for Antivenin Products (#906) and human Immune Globulin Products (#743). See also Antivenin (Crotalidae) Polyvalent (equine origin) (#907), a product essentially being replaced by CroFab.

Description: Crotalidae Polyvalent Immune Fab (Ovine) or CroFab is a purified, lyophilized (freeze-dried) antivenin formulation combining enzymatically-derived ovine (sheep) immunoglobulin Fab fragments obtained from the blood of healthy sheep immunized with one of the following North American snake venoms: Crotalus atrox (Western Diamondback rattlesnake), Crotalus adamanteus (Eastern Diamondback rattlesnake), Crotalus scutulatus (Mojave rattlesnake), and Agkis-trodon piscivorus (Cottonmouth or Water Moccasin). The four different monovalent antivenin Fab fragments are mixed in the final product.

Each monospecific antivenin is prepared by fractionation of immunoglobulin from the blood serum of venom-vaccinated sheep, partial digestion of the immunoglobulin with the enzyme papain to obtain the Fab portions of the immunoglobulin (antibody) molecules, and isolation of the venom-specific Fab fragments using ion exchange and affinity chromatography columns. Papain is used to cleave the whole antibody into Fab and Fc fragments, and trace amounts of papain or inactivated papain residues may be present in CroFab.

CroFab is a polyclonal antibody fragment product used to neutralize the poisonous effects of bites from North American crotalids (pit vipers), a family which includes the more common poisonous snakes in the U.S. – rattlesnakes, copperheads and cottonmouths. CroFab is the first new commercial antidote for rattlesnake venom in nearly half a century, and claims advantages over the only other product previously available – Antivenin (Crotalidae) Polyvalent (equine origin) from Wyeth (being discontinued), involving immunoglobulin from venon-vaccinated horses. The polyclonal Fab fragments of CroFab are smaller than the whole immunoglobulin molecule and can more quickly leave the bloodstream after injection and diffuse into tissues to neutralize the toxins against which the Fab fragments are targeted. The Fab fragment:toxin complexes formed after toxin neutralization are then rapidly cleared from the body. In vitro studies have shown CroFab to be on average five times more potent than comparable Wyeth anti-venom.

CroFab is standardized by assay of its ability to neutralize the lethal action of each of the four venom immunogens following intravenous injection in mice. One neutralizing unit is the amount of the mixed monospecific Fab protein fragments necessary to neutralize one LD50 of each of the four venoms. The potency of CroFab varies from batch to batch. However, greater than the minimum number of mouse LD50 neutralizing units against each of the four venoms shown to have efficacy in humans is included in each vial of final product.

Most snake antivenoms in use worldwide are equine (horse) derived polyclonal antibody products, including the only other product available in the U.S. (from Wyeth; being phased out). Since horse immunoglobulins are frequently associated with acute and delayed toxicity problems, physicians are sometimes reluctant to prescribe them. Besides the risk of anaphylaxis, equine immune globulin from horses vaccinated with snake venoms contains highly glycosylated immunoglobulin G(T) [IgG(T)], strongly associated with immunogenicity. Ovine (sheep)-derived polyclonal antibodies are known to produce a reduced allergic response in humans. Protherics’ manufacturing process purifies the final product to further improve safety by separating the Fab antibody fragments, discarding those antibody fragments, e.g., Fc regions, known to produce allergic responses and other side-effects, followed by affinity purification. The smaller size of the remaining, beneficial Fab fragments ensures their rapid and uniform distribution throughout the body.

CroFab is packaged in vials for intravenous administration after reconstitution with 10 mL of Sterile Water for Injection USP. Each vial of CroFab contains up to 1 g of total protein and sodium phosphate buffer, consisting of dibasic sodium phosphate USP and sodium chloride USP, with a maximum of 0.11 mg of mercury (from thimerosal, an antimicrobial preservative), and not less than the indicated number of mouse LD50 neutralizing units: Crotalus atrox (Western Diamondback rattlesnake), 1350; Crotalus adamanteus (Eastern Diamondback rattlesnake), 800; Crotalus scutulatus (Mojave rattlesnake), 5210; and Agkistrodon piscivorus (Cottonmouth or Water Moccasin), 460. The specified minimum potency per vial of clinical reference batches (and 95% reference interval) for each antivenin are Crotalus atrox, 935 (760-1,200); Crotalus adamanteus, 1,805 (1,320-2,250); Crotalus scutulatus, 11,630 (8,000-17,860); and Agkistrodon piscivorus, 755 (580-980). Each box contains 2 vials of CroFab (diluent not included). The contents of the reconstituted vials should be further diluted in 250 mL of 0.9% Sodium Chloride USP, and infused within 4 hours.

Thimerosal (see entry #939), a mercury-based antimicrobial preservative, is used as a preservative in the manufacturing process, and some residual mercury is carried over in the final product at an amount no greater than 104.5 µg per vial or approximately 0.11 mg of mercury (as ethyl mercury) per vial. This translates to a total exposure of not more than 1.9 mg of mercury per dose based on a typical total dose of 18 vials.

CroFab should be stored at 2-8°C (36 to 46˚F). The dating period when stored at 2-8˚C (refrigerated) is 30 months from the date of manufacture, fefined as the date of the initial sterile filtration of the formulated bulk.

When tested in a mouse model against a panel of 14 crotalid venoms, CroFab was an average 5.2 times (range 3.0-11.7) more potent than Wyeth’s equine antivenom.

Nomenclature: Antivenin (Crotalidae) Fab [BIO]; CroFab [TR]; CroTAb [TR former]; Crotalidae Polyvalent Immune Fab (Ovine) [FDA]; rattlesnake antivenin [SY]; pit viper antivenin [SY]; North American crotalid antivenom [SY]; Crotalus adamanteus antivenin [SY]; Crotalus scutu-latus antivenin [SY]; Crotalus atrox antivenin [SY]; Agkis-trodon piscivorus antivenin [SY]

Biological.: The most important of the immunoglobulins, IgG, comprises two heavy and two light chains with the former being coupled at their hinge region by disulfide linkages. [See the Monoclonal Antibodies entry (#300) for further antibody structural information]. Enzymatic cleavage with papain above these linkages releases two fragments capable of binding target antigens, the Fab and a crystalline fragment (Fc). Each Fab fragment contains both a light chain and part of a heavy chain, and includes the sequences responsible for specific binding to a target antigenic epitope/immunogen. The Fc, consisting of the remainder of the two heavy chains, is the site to which complement, macrophages and polymorphonuclear white blood cells can bind. Elimination of the Fc portion/component decreases related immune responses to the immunoglobulin and related adverse effects.

CroFab contains venom-specific Fab fragments of ovine immunoglobulin G (IgG) that bind and neutralize the venom toxins for which they have specificity, facilitating their elimination from the body. CroFab was effective in neutralizing the venoms of 10 clinically important North American crotalid snakes in a murine lethality model. Based on the data from this study in mice, CroFab has relatively good cross-protection against various Crotalus and other snake venoms not used in the immunization of the sheep flocks used to produce it, except for Crotalus v. helleri, where a very high dose is required, and for Crotalus molossus, where a moderately high dose is required. Preliminary data from experiments in mice using whole IgG from the sheep immunized for CroFab production suggest that CroFab might possess antigenic cross-reactivity against the venoms of some Middle Eastern and North African snakes (however, there are no confirm clinical studies to confirm these findings).

Companies.: CroFab was developed and is manufactured by Protherics plc. The FDA approval letter grants approval to Protherics Inc., CBER/FDA est. no. 1577, the U.S. subsidiary (previously Therapeutic Antibodies Inc.), with the product manufactured by the parent company in the U.K. Prior to approval of CroFab, the company had already developed and markets a comparable European Crotalidae ovine Fab product in European countries.

Filling and lyophilization (freeze-drying) services have been provided by Mayne Pharma Ltd. (Australia), now a subsidiary of Hospira. A second fill and lyophilation contractor, Chesapeake Biological Laboratories (CBL), a subsidiary of Cangene Corp., was approved by FDA in 2004.

Altana, Inc. (later Nycomed USA Inc.) originally had exclusive rights to market CroFab (and other Protherics products in development) in the U.S. Altana then had a ~100-person hospital sales force. Altana agreed to pay up to $23 million for the rights to the company’s emergency medicine products. Protherics received a royalty of 50% of net sales. The Savage Laboratories subsidiary of Altana Inc. launched CroFab in the U.S. in early Feb. 2001. Rights for CroFab (and also DigiFab) revert to Protherics in about seven years.

In 2008, BTG plc acquired Protherics.

In Aug. 2010, BTG regained U.S. marketing rights from Nycomed. BTG began U.S. sales in Oct. BTG now has exclusive marketing rights.

Manufacture: Based on information in U.S. patent 5,733,742 (see the Tech. transfer section), preparation of the immunoglobulin Fab fragments from blood in a closed, sterile environment involves (1) immunizing the animals, (2) bleeding the animals aseptically into a sterile, pyrogen-free facility, (3) contacting extracted whole blood with papain and (4) removing cellular components and much of the Fc.

Once adequate circulating antibody levels of at least 1 g/L, more preferably 3 g/L or more, are achieved following immunization, the animals are bled through a sterile needle and sterile tube into a sterile container. Typically 500 ml of blood is collected (~10 mL of blood/kg of body weight). The sterile collection container contains papain. The amount of papain used to digest whole blood varies according to the purity and specific activity of the papain. Between 0.5-2.5 g of Merck 6000 USP-U/mg papain is used per 100 ml of blood, most preferably 1.5 g/100 mL, and ethylene diamine tetraacetic acid (EDTA), disodium salt are used in combination with papain in order to enhance the activity of the papain and to chelate inhibitors of papain, respectively. L-cysteine is added at a concentration of about 1 g/100 mL blood. Ethylene-dia-minetetraacetic acid (EDTA), disodium salt, is added at a concentration of about 1.5 g/100 ml of whole blood. Papain digestion is usually carried out for between 3-5 hours at a temperature of 37˚C at pH 7.5. Papain is inactivated at the end of the digestion period by the addition of iodoacetamide, a cysteine proteinase inhibitor.

Alternatively (and, apparently, the method actually used), the papain enzyme is immobilized on a column chromatography support/matrix, e.g., cellulose (or agarose, or Sepharose), with the matrix chemically cross-linked (covalently linked) to papain (e.g., using cyanogen bromide). This solid support is placed in a centrifuge tube, the IgG-containing material is applied, the tube is centrifuged, and digested material is eluted. The immobilized enzyme solid support can be washed and reused with further amounts of IgG-containing material. Using this method, the use of a potentially toxic enzyme inhibitor to stop the enzymatic reaction can be avoided, as the immobilized enzyme can be simply removed physically.

Cellular components of the digested ovine whole blood are removed by centrifugation. At this stage, the remaining mixture contains (1) specific Fab directed against the immunogen of interest (the desired product); (2) non-specific Fab directed against numerous other epitopes (of no interest); (3) all other serum proteins (including ovine albumin) and other contaminants; and (4) inactivated papain.

The mixture is purified by ion exchange chromatography and an immune affinity purification step involving use of the specific snake venoms or venom fragments immobilized on a chromatography column (e.g., Sepharose 4B from Amer-sham Pharmacia). The Fab solution is recirculated through the column, e.g., for 18 hours at room temperature, during which time the matrix-bound venom antigen ligands bind venom-specific Fab fragments, while the remainder, up to 95% of the total Fab fragment present (i.e., ovine Fab fragments lacking specificity for the target venom), pass through and are discarded. The desired purified Fab fragments are eluted from the chromatography matrix, e.g., by washing the column with glycine buffer containing sodium chloride (0.9%), and lyophilized. This process reduces the effective amount of proteins to be used in a therapeutic dose by as much as 95%. This specific affinity chromatography step eliminates Fab fragments and antibody to irrelevant epitopes, contaminating proteins (such as albumin and papain), and other potentially toxic molecules. This approach is considered much superior to the use of non-specific affinity chromatography, e.g., with Proteins A or G, which bind essentially all antibodies.

Protherics reports it is working on a new CroFab manufacturing process designed to improve yields and considerably enhance plant throughput. FDA approval is expected towards the end of 2007.

FDA class: Biologic BLA

CBER class: Antitoxins, Antivenins, Enzymes and Venoms

Approvals: Date = 20001002; first approval, BLA (no. STN 103788/0) granted to Protherics Inc.; orphan designation (expires 10/2/2007)

Date = 20040000 (date uncertain); Indication = approval of a second filling and freeze drying contractor, Chesapeake Biological Laboratories (CBL)

Indications: = [full text of "INDICATIONS AND USAGE” section of product insert/labeling]:

CroFab is indicated for the management of patients with minimal or moderate North American rattlesnake envenomation (see Table 3 in Clinical Studies section for definitions). Early use of CroFab (within 6 hours of snakebite) is advised to prevent clinical deterioration and the occurrence of systemic coagulation abnormalities.

Status: The BLA was filed on April 27, 1998; accepted in Oct. 1999, and approved on Oct. 2, 2000, with orphan designation; approval time = ~2.45 years.

As conditions of FDA approval, Protherics committed to conducting a Phase IV post-marketing study to monitor a surrogate clinical endpoint to determine whether it can be used as a quality control parameter to detect changes in product quality that are clinically important. If appropriate, Protherics committed to evaluate results for commercial batches in a subsequent monitoring phase of the study. Protherics also committed to reevaluating the batch results for impurities in the final product (papain, albumin, and mercury) for a period of one year. This will be used to determine if production lots consistently contain these impurities significantly lower than the granted product specifications. In that case, specifications will be made more stringent (i.e., tightened).

Thimerosal (mercury-based antimicrobial preservative; see entry #939) is not added as a preservative (unlike the Wyeth product), but thimerosal is used during manufacture to assure that the affinity columns used in purification do not become bacterially contaminated with repeated use. Prior to approval, CBER asked whether alternative in-process preservatives could be used. This would have required new validation studies and would have delayed approval. Because the Wyeth product is no longer being manufactured, antivenom shortages were likely to occur, if approval was significantly delayed. Thus, CBER allowed not more than 104.5 µg mercury/vial, with a recommended maximum dose of 18 vials. A patient receiving this product would receive about 1/3 the total mercury dose of the comparable Wyeth antivenom.

Tech. transfer: U.S. patent 5,733,742, “Production of antibody fragments from whole blood,” Landon, J., issued March 31, 1998, assigned to Therapeutic Antibodies Inc. (now Protherics), describes methods of preparing polyclonal immunoglobulin Fab fragments by enzymatic cleavage of immunoglobulin molecules in blood (or serum or plasma) in a closed, sterile environment. The exemplary claim (no. 1) is “A method of preparing polyclonal immunoglobulin Fab fragments, said method comprising the following steps: obtaining whole blood from an animal under aseptic conditions; contacting said whole blood with papain or chymo-papain to digest immunoglobulins; removing cellular components from the resulting solution; and separating the resulting Fab fragments from the solution.” The papain (or chymopapain) enzyme is generally immobilized and separated on an affinity chromatography matrix using the matrix-bound snake venoms as the ligands. Classically, the production of Fab fragments from immunoglobulin and antibody preparations has relied on papain digestion. These digestion procedures have involved incubation of a solution of papain with a substantially pure preparation of immunoglobulins under defined conditions of pH, temperature and co-factors, such as cysteine and EDTA, which serve to activate the enzyme and bind its inhibitors, respectively. However, the application of papain digestion to blood, serum or plasma had never before been described. Examples in the patent cite generation and purification of Fab specific for nortriptyline, an antidepressant drug, and tumor necrosis factor (TNF).

Trials: CroFab clinical trials included the first controlled study of safety and efficacy with a Crotalid antivenin. Trials conducted at eight U.S. centers were completed in 1997. Two pivotal studies, conducted in collaboration with the Rocky Mountain Poison and Drug Center (Denver, CO), demonstrated the safety and efficacy of CroFab for the treatment of minimal and moderate N. American crotalid enveno-mations, and suggested an increased benefit to risk ratio in comparison with equine antivenin from Wyeth. A small case series from these clinical studies published in the Annals of Emergency Medicine in July 1997, concluded: “Our patients experienced a reversal of myokymic (neuromuscular) symptoms within minutes of infusion of the investigational antivenom. The administration of a new polyspecific crotalid antivenom made from ovine Fab (CroTAb) was successful in immediately and completely reversing neurotoxicity in each of these patients.”

Protherics is conducting studies to better demonstrate the benefits of using CroFab in milder Copperhead bites (to broaden the market for CroFab).

Medical: Antivenin dosage requirements are contingent upon an individual patient’s response; however, based on clinical experience with CroFab, the recommended initial dose is 4 to 6 vials. If initial control is not achieved by the first dose, e.g., after an hour, an additional dose of 4-6 vials should be repeated until initial control of the envenomation syndrome has been achieved. After initial control has been established, an additional 2-vial doses every 6 hours for up to 18 hours (3 doses) are recommended. Additional 2-vial doses may be administered as necessary, based on the patient’s clinical course.

Market: About 8,000 pit viper bites are reported in the U.S. each year. The Protherics 2006 annual report states, “We believe that CroFab has captured more than half of a potential $70–80 million market opportunity, and net revenues in the US are shared equally with our distributor, Fougera.”

Total 2006 CroFab revenues reported by Protherics were £14.1 ($29.0 million, on 7/23/2007); £11.5 million ($23.7 million, on 7/23/2007) in 2005; £13.2 million (then ~$23.16 million) in FY2004; and £8.5 (then ~$14.92 million) in 2003.

With Wyeth having discontinued its comparable product, CroFab has no competition (in the U.S. market). Protherics is working to expand the product’s U.S. market into states less familiar with its use. Protherics expects to have adequate product supplies to meet growing demand for CroFab in FY2006.

The 2007 Average Wholesale Price (AWP) is ($2,851.00/packagee containing 2 vials ($2,416.00 in 2005; $2,279 in 2004) (Red Book, 2007).

At the AWP, the recommended starting dosage of 4-6 vials would cost ~$11,400-$17,100.