(M1330-10-58) A Multiple Component, Indirect Analysis of Ammonium: Analysis of Sulfate Ion in a Commercial Biotherapeutic via HPLC to Measure Ammonium Content

Purpose: Common contaminants found in biotherapeutics are individual ions that are residuals from a specific step in the manufacturing process. Current methods for measuring ions include ion selective electrodes (ISEs), enzymatic assays, ion chromatography and colorimetric assays. Unfortunately, these assays are associated with unique challenges that limit their use in more complex systems. ISEs are complicated by cross ion interferences; enzyme-based methods are at the mercy of matrix pH, ionic strength, and additional sample preparation; ion chromatography can be costly especially when the need is only one or two analytes; and common excipients in biotherapeutics often interfere with colorimetric assays. Furthermore, most HPLC-based methods are only useful for water analysis as they lack the sensitivity to evaluate ions in more complex matrices because these molecules lack a chromophore making UV detection impractical through traditional HPLC techniques. These challenges often lead to wider precision and accuracy criteria with decreased sensitivity during validation. Ammonium sulfate is a common source of contaminating ions in biotherapeutics. The ammonium ion can cause unwanted changes in pH during manufacturing of drug products and drug substances, and high levels are potentially toxic to patients and the environment. The purpose of this study was to develop a robust and cost-effective method for the determination of ammonium ions in complex biological mixtures.  An IEX-HPLC method was developed to determine the amount of ammonium ion present as a function of the amount of sulfate in a commercial biotherapeutic when no other sources of ammonium ion have been introduced during the manufacturing process. The sulfate ion elutes as a single peak with exceptional resolution and quantitation down to 10 ppm sulfate. The sulfate content is proportional to the ammonium ion content, which was verified by historical data acquired using an analyzer method. Precision, accuracy, sample handling and preparation are compared to a conventionally used enzymatic method from literature. With additional development, this method has also shown the capability to evaluate relative amounts of sodium depending on the sample matrix making the basis of a multicomponent assay.

Methods: The drug substance (DS) is directly placed into centrifuge filters to remove a substantial proportion of the protein. The filters are centrifuged for 15 minutes and the flow through transferred to HPLC vials. Following injection of the samples, a step gradient is run to separate the remaining matrix components and protein from the ions. The eluted ions are detected by a charged aerosol detector (CAD) and the peak areas quantified. The peak areas are compared to sulfate ion standards to determine the amount of sulfate, and hence ammonium ion present. Ammonium ion concentrations are compared to the amount of ammonium ion determined using a conventional enzymatic assay.

Results: The IEX-HPLC method has excellent specifications and is validated according to ICH Q2(R2). The sample preparation is simple and robust. The precision of the method is less than 10% RSD within and between runs. Its accuracy was observed to be 80-120% recovery. Its sensitivity is exceptional with an LOD of 5ppm and an LOQ of 10 ppm. The range of values can accommodate most samples without dilution and the run times can accommodate a large number of samples per day. Samples were analyzed using the IEX-HPLC method and were compared to historical ammonium concentration data acquired using an analyzer. Recovery was seen as suitable in all lots of DS apart from DS Lot#4 in which the data was near the LLOQ of both methods; for this sample the IEX-HPLC performed consistently within the method limitations of the HPLC and exceeded the capabilities of the analyzer. In all instances, the measured sulfate concentration correlated to ammonium concentration which matched with expected values of the analyzer method.

Conclusion: The IEX method exhibits process improvements, tighter specifications and cost savings over other traditional methods of ion determination and quantitation. Additionally, the need for specialized equipment, or reagents per analyte is removed. In comparison experiments, suitable recovery supported excellent range and sensitivity. This HPLC method utilizing CAD can identify different ions in a simple matrix, and indirectly analyze for ammonium ions in a commercial drug substance. The promising results demonstrated from this IEX method make the analysis of ionic contaminants more practical, efficient, and robust by removing method specific challenges and open the door for simpler analysis of such components.

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