Synthesis, Chromatographic Purification, and Isolation of Epothilone–Folic Acid Conjugate BMS-753493

Abstract

We describe the synthesis, chromatographic purification, and isolation of the epothiloneÀfolic acid conjugate BMS-753493, an investigational new drug candidate for the treatment of cancer. The main challenges for process development were the instability of BMS-753493 in aqueous solution, the design and optimization of the preparative chromatography, and the removal of phosphate salts and water from the purified material. The operating conditions of the batch chromatographic purification were optimized using a column adsorption model. The free-salt active pharmaceutical ingredient was isolated via the precipitation of its zwitterion following a careful determination of the isolation parameters that controlled thermal and pH-related decomposition. This process enabled the manufacturing of several batches (10À30 g) of cGMP quality BMS-753493.

Endotoxin Removal from a Small-Molecule Aqueous Drug Substance Using Ultrafiltration: A Case Study

“…The time interval during which the API solution remains stable, defined as the time at which the API HPLC purity is 0.5 area percent (AP) lower than its initial purity, is ~12 h at room temperature and at least 24 h at 5 °C. [6] Neither the API potency nor the impurity profile of the batch to be remediated showed any significant degradation at 5 °C over 16 h (Table 1c). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10 To evaluate the feasibility of the rework procedure on scale, two endotoxin-contaminated API solutions were processed using one polyethersulfone 10-kDa membrane (50 cm 2 ).…”

“…• disposable nonsterile (i.e., non gamma irradiated) 6 polypropylene containers were used to hold the HPLC mobile phase and (pre-and post-HPLC) API in-process samples.…”

“…5 Demineralized water was used in the parts washer. 6 API stability in contact with gamma-irradiated polymers was not characterized. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18…”

“…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 3 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.…”

“…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.…”