Jeffrey S. Rohrer

Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases

We developed a set of prototype cation-exchange column packings that are based on a hydrophilic coated, pellicular polymeric support with a grafted tentacular surface chemistry that is highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character and (3) have moderate loading capacity. Cytochrome c variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the presence of lysine at the C terminus of the heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

Determination of trace level perchlorate in drinking water and ground water by ion chromatography

Ammonium perchlorate, a key ingredient in solid rocket propellants, has recently been found in ground and surface waters in the USA in a number of states, including California, Nevada, Utah, and West Virginia. Perchlorate poses a health risk and preliminary data from the US Environmental Protection Agency reports that exposure to less than 4-18 micrograms/l provides adequate human health protection. An ion chromatographic method was developed for the determination of low microgram/l levels of perchlorate in drinking and ground waters based on a Dionex IonPac AS11 column, a 100 mM hydroxide eluent, large loop (1000 microliters) injection, and suppressed conductivity detection. The method is free of interferences from common anions, linear in the range of 2.5-100 micrograms/l, and quantitative recoveries were obtained for low microgram/l levels of perchlorate in spiked drinking and ground water samples. The method detection limit of 0.3 microgram/l permits quantification of perchlorate below the levels which ensure adequate health protection. A new polarizable anion analysis column, the IonPac AS16, and its potential applicability for this analysis is also discussed.

Improved method for the determination of trace perchlorate in ground and drinking waters by ion chromatography.

Ammonium perchlorate, a key ingredient in solid rocket propellants, has been found in ground and surface waters in a number of U.S. states, and perchlorate contamination of public drinking water wells is now a serious problem in California. Perchlorate poses a health risk and preliminary data from the U.S. EPA reports that exposure to less than 4-18 microg/l provides adequate human health protection. An improved ion chromatographic method was developed for the determination of low microg/l levels of perchlorate in ground and drinking waters based on a Dionex IonPac AS16 column, an hydroxide eluent generated using an EG40 automated eluent generator, large loop (1000 microl) injection, and suppressed conductivity detection. The method is free of interferences from common inorganic anions, linear over the range of 2-100 microg/l perchlorate, and quantitative recoveries are obtained for low microg/l levels of perchlorate in spiked ground and drinking water samples. The MDL of 150 ng/l permits quantification of perchlorate below the levels that ensure adequate health protection.

Determination of hexavalent chromium at the level of the California Public Health Goal by ion chromatography

Chromium is a primary drinking water contaminant in the USA with hexavalent chromium, Cr(VI), being the most toxic form of the metal. As a required step in developing a revised state drinking water standard for chromium, the California Department of Health Services recently issued a new Public Health Goal (PHG) of 2.5 microg/l for total chromium and 0.2 microg/l for Cr(VI). Hexavalent chromium can be determined (as chromate) by ion chromatography, as described in US Evironmental Protection Agency Method 218.6; however, the method as originally published does not allow sufficient sensitivity for analysis at the California PHG level of 0.2 microg/l. Modification of the conditions described in Method 218.6, including the use of a lower eluent flow-rate, larger reaction coil, and larger injection volume, significantly increases the method sensitivity. The modified method, which uses IonPac NG1 and AS7 guard and analytical columns, an eluent of 250 mM ammonium sulfate-100 mM ammonium hydroxide operated at 1.0 ml/min, a 1000 microl injection volume, and postcolumn reaction with 2 mM diphenylcarbazide-10% methanol-0.5 M sulfuric acid (using a 750 microl reaction coil) followed by UV-Vis detection at 530 nm, permits a method detection limit for chromate of 0.02 microg/l. This results in a quantitation limit of 0.06 microg/l, which is more than sufficient for analysis at the California PHG level. Calibration is linear over the range of 0.1-10 microg/l and quantitative recoveries (>80%) are obtained for chromate spiked at 0.2 microg/l in drinking water. The modified method provides acceptable performance, in terms of chromate peak shape and recovery, in the presence of up to 1000 mg/l chloride or 2000 mg/l sulfate.

Determination of trace anions in concentrated weak acids by ion chromatography.

Ion-exclusion chromatography (ICE) followed by ion chromatography (IC) was used for the determination of trace anionic contaminants in concentrated weak acids. The ICE separation was used as a pretreatment step to isolate the contaminant anions of strong acid from the excess of matrix ions. Then a fraction containing the analyte ions was separated using IC with suppressed conductivity detection. Microbore-ion-exchange columns were chosen to address the increased purity requirements for use of these concentrated acids in semiconductor applications. The chromatographic conditions were optimized for determining trace chloride, sulfate, phosphate, and nitrate in concentrated 24.5% (v/v) hydrofluoric acid; trace chloride, sulfate, and nitrate in concentrated 85% (w/w) phosphoric acid and trace chloride and sulfate in concentrated 0.7% (v/v) glycolic acid. Method detection limits for the anions of interest were below 100 micrograms/l.

Protein Variant Separations Using Cation Exchange Chromatography on Grafted, Polymeric Stationary Phases

We developed a set of cation exchange column packings (ProPac™ WCX‐10 and ProPac™ SCX‐10) that are based on a hydrophilic coated, pellicular polymeric support grafted with polymer chains bearing ion exchange functionalities. The supports are highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character, and (3) have moderate loading capacity. Cytochrome C variants (bovine, horse, rabbit) were baseline‐separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the number of lysine residues at the C terminus of their heavy chains were baseline‐resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

Direct determination of tryptophan using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.

Determination of trace anions in high-nitrate matrices by ion chromatography.

An ion chromatography method was developed to determine trace anionic contamination in matrices that have a high concentration of nitrate ion. Contaminant anions of interest were separated on an IonPac AS15 high-capacity anion-exchange column and detected by suppressed conductivity detection. An EG40 eluent generator was used to prepare high-purity and carbonate-free potassium hydroxide. Using the EG40, performance at trace levels was enhanced because background conductivity decreased and retention time reproducibility improved. Trace anionic contamination from the mobile phase was minimized when using the eluent generator compared to using conventionally prepared sodium hydroxide eluents. The signal-to-noise ratio was also improved with the use of a temperature controlled conductivity cell and chromatography hardware in the microbore (2-mm) format. The eluent concentration was optimized to separate the contaminant anions from the excess of the nitrate matrix ions. The procedure was demonstrated for a solution of reagent-grade sodium nitrate and high-purity 0.7% nitric acid. Method detection limits for chloride, sulfate and phosphate of 150 microg/l and lower were achieved.

Identification of tobramycin impurities for quality control process monitoring using high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

Commercial-scale fermentation for tobramycin manufacture is carried out with Streptomyces tenebrarius. Impurity profiling during various phases of pharmaceutical production is important for evaluating the effectiveness of a processing step and meeting regulatory requirements. High-performance anion-exchange (HPAE) chromatography with integrated pulsed amperometric detection (HPAE-IPAD) is a highly sensitive method used to assay tobramycin and to assess purity, but no prior publications demonstrated the capability of this technique to monitor purity at various stages of production at either the typical concentrations or in the typical matrices of a manufacturing process. In addition, the identities of the impurity peaks observed in commercial sources of tobramycin when assayed by using HPAE-IPAD are mainly unknown. Regulatory agencies generally require these impurities to be characterized when found above certain limits, and when present at higher levels require toxicological studies. In this paper, we analyze tobramycin samples using HPAE-IPAD at different stages of production and show the impurity profile and concentration changes through the manufacturing process. We successfully identified nearly all the impurity peaks found in commercially available tobramycin, based on known degradation pathways deduced from extreme pH forced degradation studies, which we experimentally reproduced, and based on previously known related substances found in S. tenebrarius fermentation broth. In crude and final tobramycin products, we identified the peaks for neamine, kanamycin B, nebramine, kanosamine, 2-deoxystreptamine. We tentatively identified deoxystreptamine-kanosaminide in crude and final products, and kanamycin A, carbamoyl-kanamycin B and carbamoyl-tobramycin in down stream process intermediates of a S. tenebrarius fermentation culture. Results presented in this paper support the effective use of the HPAE-IPAD method for in-process impurity profiling of tobramycin, and as a stability-indicating technique after product purification.

Determination of magnesium and calcium in 30% sodium chloride by ion chromatography with on-line matrix elimination

Abstract Ion chromatography with on-line matrix elimination reliably determines low microgram-per-litre amounts of magnesium and calcium in brine (30% NaCl). Magnesium and calcium from 100 μl of brine were concentrated on a metal-chelating resin containing iminodiacetate functional groups. After excess sodium was rinsed away, magnesium and calcium were eluted from the metal-chelating column, separated on a cation-exchange column, and detected by suppressed conductivity. This method was reproducible at 5 μg/l magnesium and calcium in brine.