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Using a Multimode Ion Source to Improve Sample Throughput in Drug Discovery Applications
Dr. Julie Marr - Agilent Technologies

In analyzing lead molecules by LC/MS, the large numbers of samples precludes optimization of methodology for each compound. Since most molecules will never make it through the process, the goal is to get meaningful results on the first injection. This work describes an ion source capable of generating ions by both electrospray and atmospheric pressure chemical ionization simultaneously over a broad HPLC flow range. By combining this source with polarity switching modes, there is a high probability of getting ions indicative of molecular weight in one injection. Using a range of pharmaceutical products, the source was evaluated for use in compound confirmation, sample purification and unknown identification by accurate mass measurement using both quadrupole and time-of-flight mass spectrometry. Since both ionization processes are occurring simultaneously, it was evaluated for high throughput applications where short run times and narrow LC peaks are produced. Since the operating range of the source was up to 2 mL/min, it was combined with short small particle size columns that are operated in this flow range. Overall system performance was tested for the ability to get the maximum information in the minimum time.

Fast and efficient monitoring of protein expression and refolding screen with an automated Lab-on-a-chip platform
Dr. Tanja Wulff - Agilent Technologies

In the areas of target validation and high throughput screening, the need for pure and correctly folded target protein is of primary importance. Furthermore, the growing use of structural biology to support medicinal chemistry efforts makes similar demands on a viable protein supply. To this end, a growing number of research organizations are employing a multiple parallel protein supply paradigm. In such approach proteins are produced, purified, and processed in titer plate format for subsequent experiments, the need for high throughput, parallel protein analytical systems is also increasing. Through the use of high throughput expression and purification systems, investigators are able to produce multiple protein targets under a variety of conditions. The automated Lab-on-a-Chip Platform used in this study addresses these new needs by providing parallel protein analysis in a reusable microfluidic format. Sizing and quantitation can be performed in an unattended fashion on titer plates. Data for the utility of this platform for protein expression, protein purification, and protein refolding optimization experiments is presented. A new result flagging software feature affords rapid, high-level review of analyses with all data from the platform database. This permits straightforward optimization of protein expression conditions, protein purification protocols, and protein refolding screens.

Session Topic: Antibody/Protein Discovery & Development

A Compliant Solution for Monitoring Proteins Using a Lab-on-a-Chip Instrument
Steve Royce - Agilent Technologies

In August 2002, the FDA announced a significant new initiative, Pharmaceutical Current Good Manufacturing Practices (CGMPs) for the 21stCentury, to enhance and modernize the regulation of pharmaceutical
manufacturing and product quality. With the increasing focus on proteins as pharmaceutical drugs, there is a strong demand for standardized and reproducible protein analysis methods that comply with the GLP, GMP and 21CFR Part 11 requirements.

We have compared chip-based protein analysis with regard to sensitivity, sizing accuracy and reproducibility to SDS-PAGE. Data were comparable to that obtained from Coomassie-stained PAGE gels. The benefits of working on the microfluidic scale include speed of analysis, sample size and fully automated data evaluation. Ten samples can be run in thirty minutes using only four microliters of sample. The data analysis is automatically performed in real-time and is stored and archived in digital format. Electropherograms plotting fluorescence intensity against separation time are generated for each sample. Data for individual constituents of a complex mixture are shown along with calculations of concentration and percent total for each protein in the trace. IQ & OQ/PV tools and services as well as 21CFR Part 11 compliant software tailor the instrument for use in regulated environments.

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Multi Mode (ESI/APCI) Source

• Improved Sensitivity and consistency of MS pulses with new
  PDF technology
• Fast, sensitive, high-throughput ionization for proteins and polymers.
• Clean, searchable MS/MS spectra at attomole levels
• Provides attomole-level sensitivity
• Powerful combination when paired with the Agilent LC/MSD Trap or TOF

• Automated & unattended sample handling
• Fast and reliable sample analysis
• Reproducible & customizable data analysis