Douglas D. Richardson

Reversed phase ion-pairing HPLC-ICP-MS for analysis of organophosphorus chemical warfare agent degradation products

The separation and analysis of three organophosphorus chemical warfare degradation products is described. Ethyl methylphosphonic acid (EMPA, the major hydrolysis product of VX), isopropyl methylphosphonic acid (IMPA, the major hydrolysis product of Sarin (GB)), and methylphosphonic acid (MPA, the final hydrolysis product of both) were the analytes and were separated by reversed phase ion-pairing high-performance liquid chromatography (RP-IP-HPLC) with the use of myristyl trimethylammonium bromide as ion-pairing reagent and an ammonium acetate–acetic acid buffer system (pH 4.85). An Agilent 7500ce inductively coupled plasma mass spectrometer (ICP-MS) equipped with collision/reaction cell technology was coupled to the chromatographic system for detection of 31P and 47PO+. Historically, ICP-MS detection of phosphorus has been limited due to its high first ionization potential (10.5 eV) and the presence of severe nitrogen polyatomic interferences (such as 14N16O1H+ and 15N16O+) overlapping its only isotope at m/z = 31. Implementation of an octopole reaction cell with helium as the cell gas allowed for removal of the nitrogen polyatomic interferences and reduction of background signal. Detection limits for EMPA, IMPA, and MPA were found to be 263, 183 and 139 pg mL−1, respectively, with separation in less than 15 min. The developed method was successfully applied to the analysis of spiked environmental water and soil samples.

Detection of chemical warfare agent degradation products in foods using liquid chromatography coupled to inductively coupled plasma mass spectrometry and electrospray ionization mass spectrometry.

The following work presents the exploration of three chromatographic separations in combination with inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of chemical warfare agent degradation products (CWADPs). The robust ionization of ICP is virtually matrix independent thus enabling the examination of sample matrices generally considered too complicated for analysis by electrospray ionization (ESI) or atmospheric pressure chemical ionization MS with little to no sample preparation. The analysis was focused on detecting CWADPs in food matrices, as they present possible vehicles for terrorist contamination. Due to the specific detection of (31)P by ICP-MS, resolution of analytes of interest from other P-containing interferences (H(3)PO(4)) was a crucial part of each separation. Up to 10 CWADPs were separated in the presence of H(3)PO(4) with detection limits in the low part per billion levels using the methods described. Additionally, one method was tailored to be compatible with both ICP-MS and ESI-MS making structural verification possible.

Screening hydrolysis products of sulfur mustard agents by high-performance liquid chromatography with inductively coupled plasma mass spectrometry detection

Sulfur mustard (HD), bis(2-chloroethyl)sulfide, is one of a class of mustard agents which are chemical warfare agents. The main chemical warfare hydrolysis degradation products of sulfur mustards are: thiodiglycol, bis(2-hydroxyethylthio)methane, 1,2-bis(2-hydroxyethylthio)ethane, 1,3-bis(2-hydroxyethylthio)propane, and 1,4-bis(2-hydroxyethylthio)butane. The aim of this study is to identify these five hydrolysis degradation products utilizing reversed-phase high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (ICP-MS) for element-specific sulfur detection using a collision/reaction cell and electrospray ionization mass spectrometry to confirm the identification. To date, this is the first study utilizing ICP-MS with 32S element-specific detection for the analysis of vesicant chemical warfare agent degradation products.

A View on the Importance of “Multi-Attribute Method” for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy

Today, we are experiencing unprecedented growth and innovation within the pharmaceutical industry. Established protein therapeutic modalities, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, are being used to treat previously unmet medical needs. Novel therapies such as bispecific T cell engagers (BiTEs), chimeric antigen T cell receptors (CARTs), siRNA, and gene therapies are paving the path towards increasingly personalized medicine. This advancement of new indications and therapeutic modalities is paralleled by development of new analytical technologies and methods that provide enhanced information content in a more efficient manner. Recently, a liquid chromatography-mass spectrometry (LC-MS) multi-attribute method (MAM) has been developed and designed for improved simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes (Rogers et al. MAbs 7(5):881–90, 2015). Based on peptide mapping principles, this powerful tool represents a true advancement in testing methodology that can be utilized not only during product characterization, formulation development, stability testing, and development of the manufacturing process, but also as a platform quality control method in dispositioning clinical materials for both innovative biotherapeutics and biosimilars.

On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

Chapter 16 HPLC-ICP-MS screening for forensic applications

Publisher Summary This chapter discusses high performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC–ICP–MS) screening for forensic applications. There is a wide range of substances, or toxins, that pose a threat to human safety. These toxins exist in the environment both naturally and man-made. Some man-made toxins are designed with destructive applications in mind (anthrax, chemical-warfare agents), others designed with good intentions are toxic to humans (pesticides), while more are byproducts from industry/manufacturing (lead from fossil fuels). Regardless of their route to the environment and consequently to humans, scientific methods for the detection of these compounds are of great importance. These methods need to be applicable in a variety of sample matrices, including environmental (water, soil, air, etc.), foods and beverages, and biological samples (hair, blood, urine, etc.). Because of the large variety of sample types, methods should be selective and able to resolve analytes from possible interferences. HPLC–ICP–MS can be applied to a large number of sample types while analyzing for numerous elements. There are several important factors to consider when choosing the speciation method. Sample preparation is crucial, as preserving the native form of the species must be balanced with extraction efficiency.

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

ABSTRACT For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.

Proof-of-concept: Interfacing the liquid sampling-atmospheric pressure glow discharge ion source with a miniature quadrupole mass spectrometer towards trace metal analysis in cell culture media

In an effort to provide a mass spectrometry system capable of at-reactor trace metal analysis of bioprocess media and clarified cell culture fluid, the liquid sampling-atmospheric pressure glow discharge (LS-APGD) ion source was interfaced with a Waters QDa mass spectrometer. The LS-APGD is capable of elemental analysis, as well as organic compound determinations. The Waters QDa is a compact, rack-mounted single quadrupole mass analyzer commonly employed in an integrated liquid chromatography system. By replacing the QDas standard electrospray ionization (ESI) source with the LS-APGD, trace metal analysis of bioprocess stock media samples can be performed, alleviating use of high-end, inductively coupled plasma (ICP-MS) instruments. Presented here is a proof-of-concept effort, interfacing the microplasma to this platform for the first time. Preliminary optimization of the LS-APGD operating parameters and the QDas in-source collision induced dissociation (CID) conditions was performed, yielding signal intensities of >6 × 107 AU for a multi-element test solution containing 25 μg mL−1 of Cu, Ag, and Tl. As further proof-of-concept, Chinese hamster ovary (CHO) cell culture medium was spiked with the same elemental concentrations and analyzed on the LS-APGD/QDa system. Stable plasma response allows spectral background subtraction of the media components, yielding analytically-relevant elemental signals. These results suggest that the LS-APGD/QDa coupling may be a viable approach for at-bioreactor, elemental analysis.