Monitoring for contaminants is a critical step during the production process in the pharmaceutical and medical device industries. Endotoxin, a frequent contaminant, can cause fever, inflammation, headache, nausea, and even death. Found in the cell walls of gram-negative bacteria, endotoxin has routinely been detected by the sensitive and specific
Limulus
amebocyte lysate (LAL) assay. In the presence of endotoxins, LAL coagulates through an enzyme-mediated cascade, which has then been traditionally quantified based on gelation and turbidity.
The Pyrochrome assay from Associates of Cape Cod, inc. (ACC) contains a synthetic substrate with paranitroaniline chromogen (pNA), which enables spectrophotometric quantitation. Clotting enzyme is (indirectly) activated by endotoxin. The enzyme cleaves the substrate, liberating the pNa, which absorbs light at 405 nm. The absorbance (A
405
) can be measured by a plate reader and is proportional to the endotoxin concentration in the sample.
Figure 1: ()
Maintaining dat...
As production facilities strive for greater efficiency, a common practice is to seek process improvements in quality control (QC) operations. All production facilities share some common requirements for these QC checks, especially that they can be fast, simple, proven, and economical. In addition to this, the production of biological materials such as proteins frequently presents additional requirements, including the need to perform QC tests using very small volumes and the ability to measure the protein product at strength without dilution. The NanoDrop 8000 was designed and tested with these criteria in mind. Traditional spectrophotometry methods are cuvette based; however, the use of cuvettes is in many ways highly undesirable. By creating a liquid column using only a 2-µl sample (Photo 1), NanoDrop spectrophotometers eliminate the need for cuvettes, circumventing the limitations of traditional spectrophotometry, including the need for dilutions.
Photo 1:
Photo 1: ()
Speed
Traditional spectrophotomete...
The complete characterization and analysis of biopharmaceuticals includes the application of size- exclusion chromatography (SEC) to measure protein aggregates and other size variants. Soluble protein aggregates in particular can contribute to immunogenicity. Accurate analysis and quantitation of biotherapeutic protein aggregates is therefore often required.
Current HPLC/silica-based SEC methods can be time- consuming and unreliable. Their uncertain results may be due to changes in retention time, peak shape, or spacing between peaks as well as irreproducibility between columns and changes in columns within a few runs. With the Waters ACQUITY UPLC® H-Class Bio System and below–2-µm ACQUITY UPLC BEH200 SEC column chemistry, SEC separations can be obtained reproducibly, reliably, and in shorter analysis time with minimal development. The ACQUITY UPLC H-Class Bio is a low- dispersion, high-pressure quaternary LC system that contains an inert flow path that, when combined with four-solvent mixing and Auto•Ble...
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