Application Notes: General

PEGylation, the process by which polyethylene glycol (PEG) chains are attached to protein and peptide drugs is a common practice in the development of biopharmaceuticals to prolong serum half-life and improve pharmacokinetics of a drug. There is increasing demand for chromatographic methods to separate the modified isoforms from the native protein. This application note describes the use of size exclusion and ion exchange chromatography for the characterization of PEGylated lysozyme.

Short analyses time and high resolution are in great demand from R&D and QC departments within the pharmaceutical industry. Sub-two micron ODS reversed phase columns have recently been introduced to meet these requirements, but these columns require an ultra-high pressure HPLC system to achieve optimum performance. TSK-GEL ODS-140HTP, 2.3mm columns from Tosoh Bioscience have been developed to offer a combination of short analyses time and high resolution separations that can be run at modest pressures, making these columns compatible with conventional HPLC instrumentation. The polylayer bonding chemistry of these columns results in highly efficient and physically stable columns when operated at high linear velocities. In addition, TSK-GEL ODS-140HTP, 2.3mm columns can be efficiently operated at pressures not exceeding 9000psi in UPLC® and other ultra-high pressure HPLC systems, as well as in traditional HPLC systems.

Synthetic fused silica capillary tubing has proven to be a vital component in many separation science techniques. Recently, it has expanded to serve an array of scientific markets. In most cases, an understanding of the internal surface chemistry is of fundamental importance.

There are many misconceptions about what it means to perform fast gas chromatography (GC) and what the term fast GC implies. Fast GC is often associated with the use of hydrogen as a carrier gas and, although this is certainly a good approach, it is not always necessary to shorten the analysis time. A second misconception is that changing column dimension results in time-consuming method development. Using high-efficiency GC columns can greatly reduce the analysis time and when coupled with the method translation software, the time spent on method development can be greatly minimized.

Viscotek has been a strong advocate of good chromatography as a prerequisite for GPC (Gel Permeation Chromatography) data accuracy. Our recent work in application development has been driven by extremely difficult samples from industrial, biopharmaceutical, and academic sources. These samples present challenges ranging from sample solubility, column adsorption as well as detection issues. This report will attempt to highlight a new approach that could be very helpful in certain advanced GPC applications.

An effective UHPLC-MS method for high throughput separation, identification and quantitation of pseudoephedrine was developed on a Hypersil GOLD™ PFP 1.9 µm, 2.1 x 100 mm column.

The use of mobile phase pH to control analyte ionization states (pH-LCâ„¢) in reversed phase HPLC separations is a highly effective way to change selectivity. The ionized species of an analyte is shown to have higher polarity (less hydrophobicity) than the neutral species, which results in a loss of expected retention for that analyte. This can be attributed to less interaction with the hydrophobic stationary phase and greater affinity with the aqueous portion of the mobile phase. Ionized species also participate in ionic interactions with exposed and activated silanols, which impact peak shape and reproducibility.

Size exclusion chromatography is a powerful tool for the characterization of molecules differing in size and molar mass. It is widely used and well known for the determination of molar mass distributions and molar mass averages. For membranes GPC-SEC is a useful characterization method as it can measure the membrane characteristic pore size distribution, average pore size and molecular cut-off significantly faster than other methods.

In the last few decades, the novel functions of polysaccharides have provided a major impetus for increasing scientific attention. Among the most promising aspects are their immunomodulatory and antitumor effects, thickening agents and stabilizer effects.

Urea is commonly used in protein purification, including large-scale purification of recombinant proteins for commercial purposes, and in recombinant protein manufacturing to denature and solubilize proteins (1). In aqueous solutions, urea degrades to cyanate and ammonium, with the maximum degradation rate occurring at neutral pHs commonly used in biological buffers (2). Cyanate is problematic in urea solutions because it carbamylates proteins, which causes unwanted modifications that can alter the protein's stability, function, and efficiency. Therefore, an accurate, sensitive method for determining cyanate in urea-containing buffers is required.

Amylose is an occasionally-branched biopolymer and, together with amylopectin, the hyper-branched component, a constituent of starch. Determination of branching in amylopectin on the basis of amyloses may be performed with the help of synthetic amyloses. Synthetic amyloses from enzymatic (phosphorolytic) reaction were checked for their linearity.

Use imaging mass spectrometry to determine the biodistribution of erlotinib (Tarceva®) in pancreatic tumors from treated and untreated mice.

Supercritical Fluid Chromatography (SFC) is quickly becoming a popular choice by chromatographers for analytical and preparative separations.

Phthalates are added to plastics to increase flexibility. Recently, concerns have been raised over the effect of phthalate exposure from plastic materials on human health.

By William Goodman, PerkinElmer, Inc.