A Method for Removal of Endotoxin from Pharmaceutical Formulation - The authors describe a simple method to remove endotoxins from highly viscous formulations. - BioPharm International

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A Method for Removal of Endotoxin from Pharmaceutical Formulation
The authors describe a simple method to remove endotoxins from highly viscous formulations.


BioPharm International
Volume 25, Issue 9, pp. 26-29

ABSTRACT

In this study, strong anion exchange membranes were found to depyrogenize highly viscous formulations (viscosities 20–35 mPas, ≥ 640 mg substance/mL) of triodinated X-ray contrast agents, such as Visipaque and the Gd-chelate based MR-contrast agent Prohance, without the need to dilute the formulation. The method was simple to perform and had no impact on pH, ionic strength, or concentration of gadolinium. The applicability of the method was demonstrated on endotoxin-contaminated formulations with different ratios of Ca 2+ and Na + , which remained unchanged after treatment with the anion membrane.

Working with new experimental drugs for in vivo studies requires careful control of the endotoxin content, so that any adverse effects caused by endotoxin are not superimposed on side effects from the investigational drug. The authors therefore developed a method that could give endotoxin values < 1 EU/mL without changing the composition of the drug formulation.

Lipopolysaccharide (LPS) is composed of a carbohydrate portion and lipid-A, and appears in solution as molecular aggregates between 10,000 and 1,000,000 Da. Lipid A contains phosphate groups that give the molecule a negative charge and allow use of an anion exchanger for removal (1). The common methods for removal of endotoxin (LPS) from solutions are ultrafiltration, adsorptive matrices such as chromatographic resins, and ion-exchange resins (2–4). The choice of method is dependent upon the concentration and type of drug molecule, the molecular weight, ionic strength, pH of the medium, pI, and the interaction between the drug molecule and LPS (i.e., aggregation). For highly viscous solutions, attempts to remove endotoxin by any of the common methods was unsuccessful because of long centrifugation times and clogging of the filters.

The authors identified a method that can be used as a final step directly on formulations of X-ray contrast agents with high viscosities (20–35 mPas, ≥ 640 mg substance/mL) and gadolinium-based magnetic resonance (MR) contrast agents (5).

Because most methods require diluted solutions, the high concentration of nonionic contrast agent provides an advantage by lowering the solubility of LPS in the medium and pushing this negatively charged species onto the exchanger. For research on new X-ray contrast agents such a method is of high value and has not been described in the literature. A search in Scifinder retrieved only one publication describing the removal of endotoxin from pharmaceutical formulations with high viscosity (6).

MATERIAL AND METHODS

Samples

Tri-iodinated aromatic X-ray compounds : Visipaque (Iodixanol 320 mg iodine/mL in 10 mM TRIS buffer pH 7.4, osmolality adjusted to 290 mmol/kg with NaCl, viscosity = 24 mPa) was used as well as several other chemically related derivatives formulated to > 320 mg I/mL having viscosities in the range 24–35 mPa.

Gd-chelates for MR : Prohance (Gd-DOTA, 0.8 M in 10 mM TRIS buffer pH 7.4, osmolality adjusted to 290 mmol/kg with NaCl) was used.

Preparation of stock solution of LPS from Escherichia Coli

One vial (10 mg, 10 e7 EU, Sigma Corp., USA) was added to 10 mL water for injection (WFI) and 1 mL acetonitrile to give a concentration of 9.1 e6 EU/mL = 9.1 e3 EU/ÁL.

Preparation of working solution of LPS from E. Coli

0.1 mL of the stock solution was diluted to 10 mL with WFI (dilution factor = 100) to give 91 EU/ÁL.

Depyrogenation procedure

Filter device: 15 cm2 Sartobind Q15 (Sartorius, NY) membranes integrated in ready-to-use units with pore size 3–5 Ám made of cellulose modified with positively charged quaternary ammonium groups.

Equilibration and sterilization of Sartobind Q15 : Each filter was eluted with 50 mL 10 mM TRIS pH 7.4, followed by 5 mL 70% ethanol that was allowed to stay in the filter for 3 min, and finally with 50 mL 10 mM TRIS pH 7.4.

Endotoxin removal : The formulations were passed through the filter units by a flow of 1–2 drops/s.


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