Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

Development of supercritical fluid extraction and supercritical fluid chromatography purification methods using rapid solubility screening with multiple solubility chambers.

Rapid solubility screening in diverse supercritical fluids (SCFs) was carried out via multiple solubility chambers with a trapping device and online ultraviolet (UV) detection. With this device, it was possible to rapidly study the solubility variations of multiple components in a mixture. Results from solubility studies have been used to develop efficient supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) methods. After the investigation of solubilities of theophylline and caffeine in several neat organic solvents and SCFs, advantages of SFE over conventional organic solvent extraction were demonstrated with a model mixture of theophylline and caffeine. The highest solubility ratio of 1:40 (theophylline:caffeine) was observed in the SCF with 20% acetonitrile (MeCN), where a ratio of 1:11 was the highest in the neat organic solvents. A model mixture of theophylline:caffeine (85:15 w/w, caffeine as an impurity) was successfully purified by SFE by leveraging the highest solubility difference. The SCF with 20% MeCN selectively removed caffeine and left theophylline largely intact. Rapid SCF solubility screening was applied to development of SFE and SFC methods in a drug discovery environment. Two successful applications were demonstrated with proprietary Amgen compounds to either remove an achiral impurity before chiral purification or enhance chiral chromatographic throughput.

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases.

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.

A systematic investigation of recovery in preparative reverse phase high performance liquid chromatography/mass spectrometry.

In this paper we report a systematic recovery study based on reversed phase high performance liquid chromatography (RP-HPLC) separation and mass spectrometric (MS) based fractionation. Factors including a compound's physicochemical properties, column mass loading and presence of impurities were investigated through commercially available compounds. Results suggest that the delay time between MS peak detection and fraction collection, fraction detector's signal-to-noise ratio and compound's base peak width in the chromatogram have the biggest impacts on purification recovery. In an effort to assess sample recovery within our high throughput purification process, re-purification was performed on four compound libraries that were synthesized in-house. Reproducible recoveries (>80%) were achieved in all tests.

Buffer system for the separation of neutral and charged small molecules using micellar electrokinetic chromatography with mass spectrometric detection.

An organic buffer system will be discussed that is suitable for the separation of neutral as well as charged molecules be means of micellar electrokinetic chromatography (MEKC). The buffers are based on the combination of a long chain alkyl acid, such as lauric acid with ammonium hydroxide or an organic base such as tris-hydroxymethylaminomethane (Tris). The resulting buffer system is able to separate neutral compounds based on its micellar properties. These buffers exhibit much reduced conductivity compared to traditional MEKC buffers, such as sodium dodecylsulfate (SDS), which contain inorganic salts. They also have inherent buffer capacity at high pH resulting from the basic buffer component, which in our studies had pK values from about 8-11. The separations that were observed showed high efficiency with plate counts in many cases above 500,000 plates per meter. The reduced conductivity allowed for the application of much higher electric fields, resulting in very fast analysis times. Alternatively, an increase in detection sensitivity could be achieved, as the reduced conductivity allowed for the use of capillaries with lager internal diameters. Combinations of different alkyl acids and organic bases provided for significant flexibility in selectivity tuning. Finally, the fact that the organic micellar buffer systems discussed here do not contain inorganic ions, allows for coupling with mass spectrometric (MS) detection. The possibility of MS detection combined with the high speed in analysis that can be obtained using these organic buffer systems, could make this approach an interesting option for high throughput analysis of combinatorial libraries.

Ammonia as a preferred additive in chiral and achiral applications of supercritical fluid chromatography for small, drug-like molecules.

Supercritical fluid chromatography is routinely utilized by analytical separations groups in the pharmaceutical industry to efficiently handle separations for discovery medicinal chemistry purposes. Purifications are performed on samples ranging from a few milligrams up to hundreds of grams. Basic additives dissolved into the liquid component of the SFC mobile phase are commonly used to improve peak shape and efficiency in achiral and chiral separations. While for purposes of analysis there is minimal consequence to additive introduction in the mobile phase, for preparative separations one needs to consider the potential effect of an additive's presence when concentrated with the desired compound. Following an SFC purification using an additive-containing modifier, the resulting fractions will contain an easily evaporated modifier, and after its evaporation perhaps still significant levels of the less volatile additive. Depending on the aqueous solubility and basicity of the final product, the process of removing basic amine additives can be time-consuming and can result in reduced yields. NMR analysis following preparative isolation and evaporation often reveals the fact of insufficient removal of the chromatographic additive even after aqueous work up steps. In this study, ammonia is evaluated as an alternative additive to strong bases such as diethylamine (DEA) in SFC purification and analysis and to the authors' knowledge no previous publication has been written describing the application of methanolic ammonia as an additive for SFC separations. Dimethylethylamine (DMEA), a more volatile additive than DEA, is also evaluated relative to ammonia for its potential to simplify the isolation process after purification and in terms of chromatographic performance. The loss in concentration of ammonia in methanol modifier over time due to evaporation and effects of that loss are also described. Furthermore, for ammonia the analytical benefit is shown to extend to on-line mass spectrometric detection relative to other basic additives.

Characterization of short-term temperature, exposure, and solubilization effects on library compound quality.

Many compound collections are stored under the same temperature conditions, which can limit flexibility by increasing the processing time required for high-demand compounds. In this study, the authors wanted to evaluate the impact of a hybrid-storage approach where high-demand compounds are stored for a shortened time period at room temperature to expedite processing operations. The use of a Covaris adaptive-focused acoustics platform was also characterized as a potential enhancement or alternative to storage at elevated temperatures. This study evaluated the impact of temperature, exposure, and solubilization on overall compound quality for short-term storage. A small library of 25 representative compounds was evaluated over an 18-week period to monitor the change in purity and concentration by high-performance liquid chromatography with ultraviolet detection. The authors concluded that temperature had a significant impact on compound concentration, and the effects due to exposure cycles were minimal. A storage time of 12 weeks at room temperature resulted in minimal compound loss, but storage times beyond this would be unacceptable because of a >20% decrease in concentration. Finally, the acoustic solubilization protocol also increased the number of compounds at the target concentration with no impact on overall purity, leading to a potential for increased storage times at frozen temperatures.

Mass spectrometry analysis of new chemical entities for pharmaceutical discovery.

In this Section, we review the applications of mass spectrometry for the analysis and purification of new chemical entities (NCEs) for pharmaceutical discovery. Since the speed of synthesis of NCEs has dramatically increased over the last few years, new high throughput analytical techniques have been developed to keep pace with the synthetic developments. In this Section, we review both novel, as well as modifications of commonly used mass spectrometry techniques that have helped increase the speed of the analytical process. Part of the review is devoted to the purification of NCEs, which has undergone significant development in recent years, and the close integral association between characterization and purification to drive high throughput operations. At the end of the Section, we review potential future directions based on promising and exciting new developments.

Enhanced performance test mix for high-throughput LC/MS analysis of pharmaceutical compounds.

LC/MS is being used for the routine analysis of small molecules in both the discovery and development stages within the pharmaceutical industry. In drug discovery, LC/MS is relied upon to confirm the identity and assess the purity of chemical entities. To ensure the quality of LC/MS analysis, it is important that the LC/MS system is operating within defined performance criteria. Performance monitoring of the system with a standard compound mix offers many advantages over other alternatives, since it monitors the LC/MS system as an integrated unit under the same working conditions as those used for the analysis of samples. It is also a convenient approach, because the test mix can be injected as part of the automated sequence. Use of a test mix for similar purposes has been described previously (Tang, L.; Fitch, W. L.; Alexander, M. S.; Dolan, J. W. Anal. Chem. 2000, 72, 5211-5218). To monitor the performance of ArQule's LC/MS operation (with UV and ELS detection) in greater detail, a set of eight compounds was selected from a collection of 137 commercially available "druglike" compounds. The compounds are generally stable and compliant with the rule-of-five criteria. This enhanced mix has a balanced selection of pKa values and covers the typical range of hydrophobicity and molecular masses of pharmaceutical compounds. Moreover, the selected compounds can generally be ionized using ESI and APCI modes with positive and negative polarity. The test mix can be used under formic acid or ammonium hydroxide conditions and with methanol or acetonitrile as an organic modifier. Performance monitoring with the enhanced mix is demonstrated with respect to ionization and mass measurement, as well as changes in gradient profile, flow rate, buffer pH, and ionic strength.

Development of a mass-directed preparative supercritical fluid chromatography purification system.

In this paper, we report the development of a mass-directed supercritical fluid chromatography (SFC) purification system. We have addressed issues on software compatibility, the interface between the preparative SFC and the mass spectrometer, and fraction collection. Good peak shape and signal were achieved in the mass spectrometry (MS) trace, allowing accurate peak detection and reliable fraction collection. Simple modifications on a commercially available fraction collector enabled fractionation at atmospheric pressure with high recovery. The SFC/MS purification system has been used in support of high-throughput library purification and has been proven to be a valuable tool in complementing our reversed-phase high-performance liquid chromatograph (RP-HPLC/MS)-based technology platform.

High-throughput microcoil NMR of compound libraries using zero-dispersion segmented flow analysis.

An automated system for loading samples into a microcoil NMR probe has been developed using segmented flow analysis. This approach enhanced 2-fold the throughput of the published direct injection and flow injection methods, improved sample utilization 3-fold, and was applicable to high-field NMR facilities with long transfer lines between the sample handler and NMR magnet. Sample volumes of 2 microL (10-30 mM, approximately 10 microg) were drawn from a 96-well microtiter plate by a sample handler, then pumped to a 0.5-microL microcoil NMR probe as a queue of closely spaced "plugs" separated by an immiscible fluorocarbon fluid. Individual sample plugs were detected by their NMR signal and automatically positioned for stopped-flow data acquisition. The sample in the NMR coil could be changed within 35 s by advancing the queue. The fluorocarbon liquid wetted the wall of the Teflon transfer line, preventing the DMSO samples from contacting the capillary wall and thus reducing sample losses to below 5% after passage through the 3-m transfer line. With a wash plug of solvent between samples, sample-to-sample carryover was <1%. Significantly, the samples did not disperse into the carrier liquid during loading or during acquisitions of several days for trace analysis. For automated high-throughput analysis using a 16-second acquisition time, spectra were recorded at a rate of 1.5 min/sample and total deuterated solvent consumption was <0.5 mL (1 US dollar) per 96-well plate.

One-minute full-gradient HPLC/UV/ELSD/MS analysis to support high-throughput parallel synthesis.

High-throughput parallel synthesis of library compounds for early drug discovery requires high-throughput analytical methods to confirm synthesis, identify reaction products, and determine purity. An ultrafast 1.0-min HPLC/UV/ELSD/MS method was developed and compared to our standard 2.5- and 5.0-min methods in order to determine if the faster method was appropriate to evaluate compound synthesis and determine purity. In addition to using standard test mixtures, a 400-member library produced by high-throughput parallel synthesis was used for comparing the various methods. Mass spectrometric detection was used for compound identification, while UV and ELSD data offered purity assessment. Compared to our longer separations, chromatographic separation achieved using the 1.0-min method was sufficient for compound evaluation and purity assessment. This ultrafast 1.0-min HPLC/UV/ELSD/MS method is expected to increase analytical throughput tremendously, provide important information faster, and reduce the overall cycle time from synthesis to screening.

A straightforward means of coupling preparative high-performance liquid chromatography and mass spectrometry.

Flow splitting to a mass spectrometer is a common way of coupling a highly specific detector to preparative or semi-preparative high-performance liquid chromatography (HPLC) purification of combinatorial libraries, drug metabolites, and characterizable impurities. The sensitive mass spectrometer consumes only a small fraction of the analyte while providing online structure-specific detection, and its output can thus be used to trigger collection of the desired fraction. Coupling mass spectrometry to preparative HPLC is difficult due to the susceptibility of the detector to fouling under conditions of high analyte concentration or solute amount, or to changes in solvent composition. We report here on a device, the mass rate attenuator (MRA), which automatically produces split ratios over a range of 100:1 to 100 000:1 under programmable user control. The MRA is a flow-control device that periodically gates a small aliquot from one liquid stream into another. The design allows the user to set the frequency of the gating without interruption of the HPLC flow stream. The MRA also allows control of the volume of the aliquot that is transferred between the flow streams. This additional control, compared to passive splitting devices, facilitates optimization of the tubing connecting the separation, detection and collection events. We demonstrate that such optimization can reduce the volume of the collected fraction without compromising recovery, thus reducing the time spent in evaporating solvents to reclaim purified products.