This work is a proof of concept of how a sequence of industrial batch separation steps together are used to form an integrated autonomous downstream process. The sequence in this case study consisted of an anion chromatography step, virus inactivation and finally a hydrophobic chromatography step. Moving from batch to integrated separation minimizes hold-up times, storage tanks, and required equipment. The conversion from batch to integrated mode is achieved by extracting operating points and separation data from batch chromatograms. The integrated separation process is realized on an ÄKTA Pure controlled by an open research software called Orbit, making it possible to operate complex process configurations including multiple steps. The results from this case study is the principle and method of the steps taken to automation, achieving a more continuous and efficient downstream process.
Monoclonal antibodies are generally produced using a generic platform approach in which several chromatographic separations assure high purity of the product. Dimerization can occur during the fermentation stage and may occur also during the downstream processing. We present here simulations in which a traditional platform approach that consist of protein A capture, followed by cation-exchange and anion-exchange chromatography for polishing is compared to a continuous platform in which dimer removal and virus inactivation are carried out on a size-exclusion column. A dimerization model that takes pH, salt concentration and the concentration of antibodies into account is combined with chromatographic models, to be able to predicted both the separation and the degree to which dimers are formed. Purification of a feed composition that contained 1% by weight of dimer and a total antibody concentration of 1 g/L was modeled using both approaches, and the amount of antibodies in the continuous platform was at least 4 times lower than in the traditional platform. The total processing time was also lower, as the cation-exchange polish could be omitted.
A simple, direct, sensitive, and selective stopped-flow method for the fluorimetric determination of uric acid in serum and urine samples is described. The variation of fluorescence intensity during the reaction between uric acid and 1,1,3-tricyano-2-amino-1-propene (triap) in the presence of hydrogen peroxide is monitored. For these kinetic measurements peroxide is monitored. For these kinetic measurements we used a versatile stopped-flow module that can be fitted to any fluorimeter. The linear range of the proposed method is 0.08-3.00 mg of uric acid per liter, and the detection limit is 0.03 mg/L. Within- and between-assay CVs and selectivity data are reported. Results for serum and urine samples correlated well with those obtained by the uricase method The proposed method is inexpensive and requires no sophisticated detection equipment.
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