Purification quality assessment

For high-throughput purification pipelines, quality assessment (QA) procedures are required which can be carried out in parallel in relatively high throughput. The QA steps routinely carried out in the OPPF are summarized in Table 2.3. Other methods that have been used in HTP projects include one and two-dimensional NMR screening and differential scanning calorimetry. 

Sample preparation (extraction, purification, and labeling) Target quality, sample matrices, extraction method, labeling technology and uniformity of dye incorporation Target quality assessments, universal reference standards, spike-in controls... 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

We typically use RNA purification by the Trizol method (Invitrogen, following the manufacturer s instructions), which has the advantage over column-based methods that it can purify small amounts of RNA and retain miRs. Purified RNA is dissolved in RJNAse-free water and stored at —80°. RNA quality is assessed on an Agilent Bioanalyzer (Agilent Technologies) or by gel electrophoresis. 

The need for highly accurate melting point determinations is rare. For quality control or routine purification assessment experiments, much less is required. Impurities equivalent to less than 1% wt/wt will result in melting point changes that can be readily observed in many compounds. 

The amount of resin to pack in a column, column geometry, flow rates, pressure, column hardware, and wetted materials of construction should all be evaluated in development. Chromatography columns must be properly packed prior to validating the purification process. From a business perspective there should be some criteria other than purification of the product by which the quality of the packed column can be assessed prior to applying the feedstream, which by this time in the process is quite expensive. Height equivalent to a theoretical plate (HETP) and asymmetry determinations can be used to evaluate the quality of column packing, but may have limited value for some types of packed columns... 

In the early days of biotechnology product development, the focus was on quality issues [4] or process-related impurities.The concerns at that time were for carryover of other cellular proteins and DNA and for contamination with endotoxins, chemicals, and viruses. Of course, these concerns still exist, but methods for purification and assays for evaluation of clearance have alleviated the need for the safety assessment scientist to focus on contaminants instead they are now asked to focus on the pharmacological activity of the molecules. An ICH guidance (Q6B Specifications Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) addresses the specific issues related to the manufacturing process [6], Other product-related issues such as impurities do need to be considered by the safety assessment scientist, for... 

The tedious purification of intermediates in classical solution synthesis produces pure compounds, which are then carried forward to the subsequent steps of the synthetic scheme. When a side product of the SP reaction remains attached to the resin, it becomes impossible to separate it from the desired intermediate/target molecule, thus irreversibly affecting the quality of the synthesis and the purity of the final product. This and other factors, which will be discussed in the following chapters, are considered during the so-called chemistry assessment phase of the synthesis in which the... 

Since application of fast, generic LC methods with mass spectrometry (LC/MS) is emerging as the technique of choice for assessing the progress and final quality of large combinatorial arrays in drug discovery, it will be discussed in some detail, along with other detection techniques. Mass-directed purification and characterization on the preparative scale will also be addressed. 

Purification, assessment of quality and storage of conjugates obtained by chemical linkage... 

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