Endotoxin Removal from a Small-Molecule Aqueous Drug Substance

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Endotoxin removal from a small-molecule aqueous drug substance using ultrafiltration: a case study Nuria de MAS, Donald C Kientzler, and Devon C Kleindienst Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00195 • Publication Date (Web): 07 Aug 2015 Downloaded from http://pubs.acs.org on August 11, 2015

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Organic Process Research & Development

Endotoxin removal from a small-molecule aqueous drug substance using ultrafiltration: a case study Nuria de Mas,1* Donald C. Kientzler,2 and Devon Kleindienst† Chemical Development and †Analytical & Bioanalytical Development. Bristol-Myers Squibb Company, One Squibb Drive, New Brunswick, New Jersey 08901, United States Abstract Endotoxin must be controlled in the manufacturing of parenteral drug products to prevent adverse reactions in patients. Endotoxin contamination may arise in the manufacturing of active pharmaceutical ingredients when aqueous solutions are present. We describe our efforts to remove endotoxin load from a small-molecule aqueous drug substance (molecular weight 1,570 Da) that was prepared by organic synthesis and purified using preparative reverse-phase HPLC. The aqueous phosphate buffer solution used in this purification step was identified as the root cause of the endotoxin contamination.

Endotoxin was removed by dead-end 10-kDa

ultrafiltration at 5 °C with excellent recovery of potency and purity. Upon meeting quality release specifications, the reworked drug substance solution was used to manufacture drug product for evaluation in clinical trials.

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Present Address: Biologics Drug Substance Manufacturing Sciences and Technology, Bristol-Myers Squibb, 35 South Street, Hopkinton, Massachusetts 01748, United States. 2 Present Affiliation: Drug Product Science and Technology, Bristol-Myers Squibb. * To whom correspondence should be addressed. E-mail: [email protected]

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Keywords: parenteral, aqueous active pharmaceutical ingredient, small molecule, endotoxin removal, ultrafiltration

Introduction The folate receptor (FR) is a cell surface receptor that is highly expressed in tumor tissues of epithelial origin while minimally expressed in normal tissues.[1] The FR binds folic acid and its conjugates tightly (dissociation constant Kd < 10-9 M). Receptor endocytosis, dissociation, and release of the conjugate inside the cell have been demonstrated in preclinical in vitro and in vivo models. Epothilones are a class of potent non-taxane microtubule stabilization agents that possess antitumor activity in cell lines that are taxane resistant, including those with overexpression of multidrug resistance proteins and β-tubulin mutations.[2, 3] BMS-753493 is an epothilone–folic acid conjugate that was designed to selectively target FR-expressing cancer cells. It showed preclinical efficacy consistent with the selective delivery of the cytotoxic epothilone into tissues that overexpress the FR and was an investigational new drug (IND) candidate for the treatment of cancer.[4, 5] The highly polar peptide fragment in compound 1 (molecular weight 1,570 Da, Figure 1) has a major influence in its physicochemical properties. The compound contains multiple ionizable groups, including four carboxylic acids, an aziridine, and a guanidine, with an isoelectric point in the range of 3.5–4.5. No crystalline forms were found through high-throughput screening of counterions (Mg2+, Zn2+, Ca2+, Tris+, K+, and others) at pH 2–9 using the antisolvents methanol, ethanol, acetone, and THF. On the other hand, the stability of its sodium salt in aqueous solution at pH 7.0 was acceptable only at subzero temperatures, with -70 °C giving the best stability over 26 weeks. As the aqueous solution was an acceptable form because 1 is administered intravenously, the sodium salt of BMS-753493 as a 10–15 mg/mL solution at pH 7.0±0.5 stored

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at -70 °C was selected as the API form for drug product manufacturing.[6, 7]

This drug

substance solution is clear, yellow to pale yellow, and essentially free from particulate matter on visual inspection. At room temperature it has modest stability with a half-life at pH 7 of 11 days and undergoes 82% degradation at pH 9.5 after 24 h. The compound 1 is purified from a crude reaction mixture by preparative reverse-phase HPLC using aqueous Na2HPO4 and acetonitrile as the mobile phases and isolated without significant degradation by converting the purified epothilone to a zwitterion and filtering to remove both buffer salts and water. Upon reconstitution of the zwitterion in aqueous solution and freezing at -70 °C, the desired API was obtained.[6] The first GMP batch of 1 met all release specifications for drug substance except for endotoxin (≤2.9 EU/mg API) (EU/mg stands for Endotoxin units/mg). This specification was set in accordance with the maximum endotoxin load allowable in drug product (≤5.8 EU/mg API), which was calculated from the maximum projected dose of the Phase I clinical trials and to allow for potential endotoxin contribution from other components of the drug product, including water and buffer salts. Endotoxin is the natural complex of lipopolysaccharides (LPS) present in the outer membrane of the bilayered Gram-negative cell. LPS is composed of a hydrophilic polysaccharide moiety that is covalently bound to a hydrophobic lipid moiety (Lipid A). LPS from most species is composed of three distinct regions: the O-antigen region, a core oligosaccharide, and Lipid A. The molar mass of an endotoxin monomer generally varies from 10 to 20 kDa depending on the oligosaccharide chain. Endotoxins exist as aggregates in aqueous solution and, in the absence of surfactants, self assemble into a variety of shapes (e.g., micelles). LPS contains exposed phosphate groups, which are negatively charged at solution pH values above 2. [8-10]

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Endotoxins are known to cause adverse reactions (immune response of varying severity) in patients upon injection. Consequently, control of endotoxin contamination in parenteral manufacturing is an important requirement that is underscored by its ubiquity in nature (particularly in aqueous environments), its remarkable stability, and the relative likelihood of its occurrence in parenteral solutions. The Limulus amoebocyte lysate (LAL) test has been the standard for the detection and quantification of endotoxin.[11] This test is based on the observation that clotting factors in horseshoe crab amoebocytes are triggered through an enzymatic cascade, including Factor C, that ends in the formation of coagulin in response to LPS as a defense mechanism against the gram-negative bacteria found in the ocean. If a clotting reaction occurs, the solution will become cloudy due to the insoluble coagulin.3 Protein therapeutics derived from bioprocesses may become contaminated with endotoxin if exposed to Gram-negative microorganisms during manufacturing. The predominant potential source of endotoxin in a pharmaceutical manufacturing process is the purified water used as a raw material. Bacteria can grow in nutrient-poor media such as water, saline, and buffers.[8, 9, 12] In contrast, the isolation of penultimate intermediates and small-molecule API on scale typically involves purification by crystallization from organic solvents, thus resulting in the virtual absence of bioburden and endotoxin load in the drug substance.

If crystallization

procedures are unsuccessful, lyophilization may be used for the removal of water from the API on laboratory scale though this isolation method is impractical for the production of bulk API. Consequently, as a general practice, endotoxin is not tracked during early small-molecule drug substance process development while conventional bioburden control and sterile drug-product manufacturing practices are sufficient to ensure the quality of the drug product manufactured from such drug substances. 3

The basis of the turbidimetric method is that this turbidity is proportional to the endotoxin concentration.

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Endotoxins are shed in large amount during cell growth, division, and death.[12] They may be found in biofilms or enter the manufacturing process from feed water or contaminated manufacturing equipment. They are highly resistant to thermal degradation and are not destroyed under regular sterilization conditions. In contrast, bioburden (i.e., viable microorganisms) may be removed by 0.22-µm filtration. Endotoxin may be inactivated when exposed to temperatures of at least 250 °C for at least 30 min or 180 °C for more than 3 h.[8, 13] Acids or bases (0.1 M NaOH in 95% ethanol)[8] may also be used to destroy endotoxin on laboratory scale. Given the importance of endotoxin control in pharmaceutical manufacturing, there have been numerous reports on processes to remove endotoxin contamination from proteins: anionexchange chromatography for positively charged proteins (as LPS is negatively charged at pH above 2 and the pH of most injectable pharmaceutical drug products is typically in the range of pH 5–7), ultrafiltration (to remove large endotoxin aggregates for small proteins), affinity adsorbents, gel filtration chromatography, sucrose gradient centrifugation, and Triton-X-114 phase separation.[8, 9] Recently, a 34-amino acid LPS-binding Sushi domain was identified in Factor C. Expression and characterization of this linear peptide showed high binding to and neutralization of LPS. This peptide is the basis of the EndoBind-R affinity chromatography columns (BioDtech, Inc., Birmingham, AL).[14] EndoBind-R contains the Sushi 3 domain of the horseshoe crab Factor C peptide bound to 4% cross-linked agarose resin and has been used to remove endotoxin from water, buffers, and cell culture media. Under optimized conditions, this method may be used to obtain endotoxin-free protein solutions with high recovery. The selection and success of these different techniques in separating LPS from the target protein is strongly dependent on the physicochemical properties of the protein. In this report, we describe the development and implementation of a procedure to remove endotoxin from the

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aqueous BMS-753493 drug substance. Endotoxin was removed by dead-end 10-kDa ultrafiltration at 5 °C with excellent recovery of API potency and purity. The obtained drug substance was employed to manufacture clinical drug product for use in Phase I clinical trials. Results and Discussion

Figure 1. BMS-753493, an epothilone–folic acid conjugate. Samples of the contaminated solution of BMS-753493 were first passed through a 0.2-µm sterile filter (polyvinylidene fluoride, PVDF) to remove bioburden. This filtration step did not change the endotoxin levels in the API solution or its potency. Two methods were screened for endotoxin removal:

adsorption onto (positively charged, quaternary amine) Mustang E

membranes and ultrafiltration using membranes with nominal molecular weight cut-offs (MWCO) of 3 kDa and 10 kDa (regenerated cellulose, YM-3 and YM-10, respectively). The evaluation criteria for both methods were endotoxin removal, as measured by the turbidimetric LAL method, and recovery of API potency, as measured by HPLC. The maximum capacity for endotoxin removal of one Mustang E syringe filter was 500,000 EU per vendor specifications. This membrane removed effectively endotoxin (

Endotoxin Removal from a Small-Molecule Aqueous Drug Substance

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