Status: approved; marketed

Organizations involved:

Genentech, Inc. – Manuf.; R&D; Tech.; World mark.

Automatic Liquid Packaging – Manuf. other

Hoffmann-La Roche Ltd. – Intl. mark.

Columbia University – Tech.; Patent dispute

Cross ref.: See also Elase ointment containing bovine DNase used for wound debridement (cleaning).

Description: Dornase alfa or Pulmozyme is an aqueous formulation of highly purified recombinant human deoxyribonuclease I (DNase I) glycoprotein expressed by Chinese hamster ovary (CHO) cells containing cDNA for the native human protein, with purification including tangential flow filtration and column chromatography. The DNase enzyme selectively cleaves deoxyribonucleic acid (DNA).

Dornase alfa is used to break down DNA that excessively thickens secretions in the lungs of cystic fibrosis patients, leading to respiratory problems and potentially deadly bacterial infections. Dornase alfa hydrolyzes the DNA in sputum of cystic fibrosis patients, reducing sputum viscoelasticity, easing symptoms and reducing infection risks. When launched in 1994, Dornase alfa was the first new therapeutic for the management of cystic fibrosis in over 30 years. Dornase alfa was the first aerosol-delivered protein to receive FDA approval.

Deoxyribonuclease I (DNase I) is an endonuclease enzyme. It splits phosphodiester linkages within polynucleotides, acting primarily on single stranded DNA (ssDNA), double stranded DNA (dsDNA), and chromatin. DNase I is activated by bivalent metal ions, e.g., Mg2+ and Ca2+. The glycoprotein contains 260 amino acids with a primary amino acid sequence identical to that of the native human enzyme. Dornase alfa has an approximate molecular weight of 37,000 Daltons (37 kDa), and a molecular formula of C1321H1995O396S9 (protein moiety).

Pulomzyme is packaged in ampules containing 2.5 ml of sterile aqueous solution containing 1.0 mg/ml (2.5 mg) dornase alfa plus 0.15 mg/mL calcium chloride dihydrate and 8.77 mg/mL sodium chloride, with a nominal pH of 6.3. The product contains no preservatives. Pulomzyme is packaged in cartons of 14 single-dose ampules in single-unit foil pouches and in 30-ampule cartons containing five foil pouches each containing 6 single-unit ampules. The antibiotic gentamicin is used during culture of CHO cells, but is not detectable in the final product.

Pulmozyme is administered by inhalation of an aerosol mist produced by a compressed air driven nebulizer system (aerosol generators; plastic tube extending from a hand-held canister). Several nebulizers are recommended for use with Dornase alfa.

Biological.: DNase is generally assayed according to the photometric method developed by Kunitz (see Kunitz, M., Journal of General Physiology, vol. 33, p. 349 and 363, 1950). One DNase unit results in an increase in absorbance at 260 nm of 0.001/minute at 25˚C when acting upon a highly polymerized solution of DNA at pH of 5.0. Also, 0.005 Kunitz unit digests 1 µg of lambda phage DNA in ten minutes at 37% in 50 mM Tris, 1 mM Mg2+, pH of 7.8 in a 50 µL reaction.

DNase enzymes are common reagents used in biochemical methods requiring digestion of DNA and recovery of RNA, or where DNA is to be removed without affecting structural proteins or enzymes. DNases are also used in tissue culture to digest DNA from damaged cells, resulting in reduced viscosity, and for removal of membrane-bound DNA fragments.

Nomenclature: DNase, rDNA [BIO]; Pulmozyme [TR]; dornase alfa [USAN BAN INN]; deoxyribonuclease (human clone 18-1 protein moiety) [CAS]; 143831-71-4 [CAS RN]; 9003-98-9 [CAS RN]; deoxyribonuclease I [SY]; E.C. 3.1.4.5 [EC]; alkaline deoxyribonuclease [SY]; DNase [SY]

Dornase alfa, Pulmozyme, and synonyms are often used ambiguously, even in technical publications, to refer to either or both the formulated product and the active ingredient.

Companies.: Dornase alfa was developed and is manufactured by Genentech, Inc. (S. San Francisco, CA), CBER/FDA est. no. 1048. The product is marketed in the U.S. by Genentech and internationally by Genentech and affiliates (including Hoffmann-La Roche Ltd., largest share-holder in Genentech).

Manufacture: A CHO cell line transfected with a recombinant plasmid containing the DNA sequence for native human DNase and adapted to production cell culture was used to prepare the Master Cell Bank (MCB). The MCB has been extensively tested and shown to be free of detectable exogenous bacterial, mycoplasmal, fungal and viral agents. The Working Cell Bank (WCB) was developed from the MCB, and also extensively tested for contamination.

Production begins with the initiation of a seed strain from cells of the WCB, which are thawed and inoculated into spinner flasks. The cell population is further expanded by serial subcultivation in vessels of increasing volume, with subsequent medium exchanges and monitoring of critical parameters including temperature, pH and dissolved oxygen. Production culture in larger vessels is carried out in serum-free medium with serial medium exchanges and tangential-flow filtration. Production medium components are added over the course of the culture in order to maintain cell viability. The medium contains the antibiotic gentamicin, 100-200 mg/L, but this is not detectable in the final product. In-process procedures are used to detect any potential contamination during this period. Standard cell culture technique is used for all sampling and product transfers. At the end of the production phase, the culture medium containing secreted dornase alfa is harvested from the culture by filtration.

Dornase alfa is recovered, concentrated, and purified from the cell culture fluid using standard chromatography (tangential flow filtration and column chromatography) and filtration methods. Purification takes advantage of the intrinsic properties of dornase alfa, e.g., hydrophobicity, charge, size. The purified protein is characterized using chemical and biochemical assays to confirm its amino acid sequence, post-translational modifications and enzyme activity. The bulk dornase alfa from purification is pooled and diluted in formulation solution to a final concentration of 1.0 mg/mL, filtered into a sterilized tank, and (at the time of original approval) shipped to Automatic Liquid Packaging (Woodstock, IL) for filling under contract. The bulk is filtered in-line prior to filling. Filling is performed using form/fill/seal technology, involving formation of 12 plastic ampules by an extrusion/vacuum molding process just prior to filling. Ampule embossing is performed concurrently with ampule formation. Filled ampules are sealed immediately in a continuation of the molding process. Filled ampules are shipped back to Genentech for placement in pouches and final packaging. The date of manufacture is the time of final sterile filtration immediately prior to filling into final molded ampules.

FDA class: Biologic PLA/ELA

CBER to CDER: Among the products transferred within FDA on June 30, 2003

Approvals: Date = 19931230, first approval; PLA ref. no. 93-0251, ELA ref. no. 93-0252; orphan designation (granted 01/16/1991; expired 12/2000); Indication = for the management of mild to moderate cystic fibrosis in conjunction with standard therapies to reduce the incidence of respiratory tract infections requiring parenteral antibiotics and to improve pulmonary function

Date = 19961202; PLA supplement; Indication = for treatment of cystic fibrosis patients with advanced disease

Date = 19980224; PLA supplement; Indication = for pediatric use in cystic fibrosis patients (infants) 3 months to 2 years, and children 2 to 4 years old

Indications: [full text of the "Indications and Use” section from product insert/labeling]:

Daily administration of PULMOZYME¨ (dornase alfa) in conjunction with standard therapies is indicated in the management of cystic fibrosis patients to improve pulmonary function. In patients with an FVC 40% of predicted, daily administration of PULMOZYME has also been shown to reduce the risk of respiratory tract infections requiring parenteral antibiotics. Safety and efficacy of daily administration have not been demonstrated in patients for longer than twelve months.

Status: The PLA received fast track review (Subpart E) and accelerated approval on the basis of improvements in surrogate markers (rather than traditional demonstration of clinical efficacy) with orphan drug designation. The review time from submission to approval was nine months (.75 year). [Granting of accelerated approval allowed Dornase alfa to become available several years earlier than would have been required to receive traditional, full, clinical endpoint-based approval].

Accelerated approval was granted on the basis of a single, double-blind, placebo-controlled, Phase III trial which demonstrated improvements in pulmonary function (FEV1) and reduction in the incidence of respiratory infections requiring antibiotic treatments among cystic fibrosis patients over five years old receiving Dornase alfa. Following approval, Genentech agreed to continue monitoring Phase III trial patients to collect long-term safety and efficacy data, to conduct a dose-ranging study in unresponsive patients, to monitor patients who had discontinued Dornase alfa treatment due to trial withdrawal-related adverse events, and to study its safety and efficacy in children less than five years old.

No centralized EU approval has been granted - approvals country-by-country.

Tech. transfer: Exemplary patents include U.S. 5,783,433, “Purified forms of DNase,” July 21, 1998, assigned to Genentech. The patent covers recombinant purified deamidated and non-deamidated human DNases, purification, and uses. The purification process involves separating deamidated and non-deamidated DNase by use of a tentacle cation exchange resin.

Genentech was a licensee of Columbia University’s patents concerning cotransformation, a broadly-useful genetic engineering method allowing selection and isolation of transformed cells. The original patents and license expired in 2000, but Columbia received another patent in 2002 and again demanded royalties, which Genentech and other companies are challenging in court. Recently, the University decided not to continue to press infringement suits and seek royalties, but the patent office is reexaming the relevant patent, and the university could against pursue infringement and royalties at a later date. See the “Tech. transfer” section of the Recombinant DNA Products entry (#100) for further information.

Medical: Cystic fibrosis (CF) occurs when individuals inherit two abnormal copies of the gene that codes for the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Heterozygotes (with one normal and one defective gene) are asymptomatic carriers, while homozygotes (two copies of the defective gene) are prone to the disease. Chronic progressive lung disease, including infections, is the major cause of death and disability in CF patients. Cyclic adenosine monophosphate (cAMP) normally regulates the chloride channel in airway epithelial cells, allowing the movement of chloride along an electrochemical gradient from the cell cytoplasm to the lumen. In CF patients, CFTR does not respond normally to cAMP and the chloride channel remains closed. This results in defective chloride secretion and airway epithelial cells show increased sodium reabsorption. The result is increased salt concentration within the lung epithelium, drawing water into airways, and dehydration of airway secretions, making them thick, tenacious, and difficult to expectorate (cough up), particularly as white blood cells (leukocytes/lymphocytes) attack bacteria in the thickened secretions. The bronchial secretions of cystic fibrosis patients contain high levels of extracellular, polyanionic DNA, which is released by disintegrating inflammatory cells (leukocytes), especially polymorphonuclear neutrophils (PMNs), present after lung infections. This DNA acts to further thicken the bronchial secretions.

DNase reduces the thickness and increases the fluidity (decreases viscosity) of airway secretions by breaking down DNA strands in sputum. CF patients typically also receive physiotherapy to reduce lung infection and inflammation, and to improve airflow and sputum (lung and airway fluids) clearance. Pulmozyme is used daily in conjunction with standard therapies for management of CF to reduce the frequency of respiratory infections requiring parenteral antibiotics and to improve pulmonary function

The Pulmozyme dose for most patients is one 2.5 mg single-unit ampule inhaled daily. Some patients may require twice-daily administration. Significant improvement in lung function generally occurs within 3- 8 days of start of therapy. Reductions in respiratory tract infections generally require several months. In patients with a forced vital capacity (FVC) greater than 40% of predicted value, daily administration of dornase alfa has been shown to reduce the frequency of respiratory infections requiring parenteral antibiotics.

Disease: Cystic fibrosis (CF) is the most common fatal genetic disease (inherited disorder) in Western countries. The median survival age for cystic fibrosis patients is 30 years. For Pulmozyme’s advanced cystic fibrosis indication, there are only about 500 cystic fibrosis patients in the U.S. with such advanced disease. Many of these patients are still in their teens and life expectancy is ~2 years for about half of these patients.

Market: Worldwide revenue was $466 million in 2009; $426 million in 2008; and $391 million in 2008. Total 2006 worldwide sales reported by Roche were CHF 436 million (~$358 million at 7/6/2007 exchange rate), up 10% from 2005 (~$325 million based on 2006 $ data); ~$353.6 million in 2004, $321.9 million in 2003, $297.2 million in 2002, $250.2 million in 2001; $226.7 million in 2000; $221.1 million in 1999; $214.0 million in 1998; $223.6 million in 1997, $218.2 million in 1996; $219.4 million in 1995; and $225.4 million in 1994. Note, swings in exchange rates can significantly affect these numbers.

Total U.S. Pulmozyme sales reported by Genentech were $223 million in 2007, $199 million in 2006, $186 million in 2005, $157.1 million in 2004, $143.7 million in 2003, $123.3 million in 2002, $110.2 million in 2001; $105.9 million in 2000; $95.8 million in 1999; $79.6 million in 1998; $78.2 million in 1997, $76.0 million in 1996; $111.3 million in 1995; and $88.3 million in 1994.

Much of the increase in Pulmozyme sales has been due to increased market penetration in the early and mild CF patient populations and the expansion of indications: to include pediatric patients.

The 2007 Average Wholesale Price (AWP) for the 2.5 mg vial is $50.38; and for 30 vials is $1,800.16 (Red Book, 2007). For comparison, the 2004 AWP was $60.01/vial; and $1,511.45 for 30 vials.

The relatively small market for CF therapeutics, only 60,000 potential users worldwide, is a major factor resulting in Pulmozyme being relatively expensive, typically costing around 12,000 euros ($15,000) per treated patient, or £5000 ($6,250) per patient per year.

In the U.S., almost all cystic fibrosis patients are considered for Pulmozyme and around 85% have used it. Median survival of U.S. patients with CF however is around seven years longer than in Europe, where Pulmozyme is used less.

In June 2006, a European comparison of six countries showed that the U.K. had the scored lowest compared to six other Euroopean countries in terms of providing access to therapeutics that help prolong lives of cystic fibrosis (CF) suffers. About two-thirds of French patients Pulmozyme, compared to fewer than one in five in the UK as a whole and only 11% in Scotland, which has the lowest life expectancy for CF patients in Europe. Many health authorities in the UK refuse to fund not only Pulmozyme but also Chiron’s TOBI (tobramycin), a highly effective but expensive antibiotic therapy for CF. Pulmozyme has not been subjected to a NICE appraisal in the U.K., resultling in a huge variation in health authorities’ attitudes to funding its use.

R&D: In Jan. 2001, citing lack of sales growth with Pulmozyme, Genentech halted development of a new version of Pulmozyme for aerosol delivery using AERx Pulmonary Drug Delivery System inhaler technology from Aradigm Corp.