Marcel Ottens

Multi‐dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

A multi‐dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion‐exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size‐exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt‐gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision‐making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge‐based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation. Biotechnol. Bioeng. 2012; 109: 3070–3083.

Robust high-throughput batch screening method in 384-well format with optical in-line resin quantification

High-throughput batch screening technologies have become an important tool in downstream process development. Although continuative miniaturization saves time and sample consumption, there is yet no screening process described in the 384-well microplate format. Several processes are established in the 96-well dimension to investigate protein-adsorbent interactions, utilizing between 6.8 and 50 μL resin per well. However, as sample consumption scales with resin volumes and throughput scales with experiments per microplate, they are limited in costs and saved time. In this work, a new method for in-well resin quantification by optical means, applicable in the 384-well format, and resin volumes as small as 0.1 μL is introduced. A HTS batch isotherm process is described, utilizing this new method in combination with optical sample volume quantification for screening of isotherm parameters in 384-well microplates. Results are qualified by confidence bounds determined by bootstrap analysis and a comprehensive Monte Carlo study of error propagation. This new approach opens the door to a variety of screening processes in the 384-well format on HTS stations, higher quality screening data and an increase in throughput.

Fourier transform assisted deconvolution of skewed peaks in complex multi-dimensional chromatograms

Lower order peak moments of individual peaks in heavily fused peak clusters can be determined by fitting peak models to the experimental data. The success of such an approach depends on two main aspects: the generation of meaningful initial estimates on the number and position of the peaks, and the choice of a suitable peak model. For the detection of meaningful peaks in multi-dimensional chromatograms, a fast data scanning algorithm was combined with prior resolution enhancement through the reduction of column and system broadening effects with the help of two-dimensional fast Fourier transforms. To capture the shape of skewed peaks in multi-dimensional chromatograms a formalism for the accurate calculation of exponentially modified Gaussian peaks, one of the most popular models for skewed peaks, was extended for direct fitting of two-dimensional data. The method is demonstrated to successfully identify and deconvolute peaks hidden in strongly fused peak clusters. Incorporation of automatic analysis and reporting of the statistics of the fitted peak parameters and calculated properties allows to easily identify in which regions of the chromatograms additional resolution is required for robust quantification.

Process Technology and Process Integration in the Preparation of Penicillins

The antibiotic penicillin G (Pen G) is the most common raw material for semisynthetic β-lactam antibiotics. Key-intermediate for β-lactam antibiotics is 6-6-aminopenicillanic acid (APA), the β-lactam nucleus, which has a worldwide annual production volume of approximately 10,000 tons. Pen G is produced fermentatively by adding phenylacetic acid (PAA) to a crude fermentation broth. Pen G is converted, either chemically or enzymatically, to APA and PAA. Coupling chemically or enzymatically different side chains to APA can yield a wide range of semisynthetic penicillins with different specificities and stabilities. Examples of bulk semisynthetic antibiotics are Amoxicillin (Amox) and Ampicillin (Ampi) (Van de Sandt and De Vroom, 2000; Bruggink et al., 1998). These antibiotics have market sales worth of ca.

Improving Gelatin Plant Productivity By Modelling The Demineralization Process Of Animal Bones

3 billion per year as bulk-formulated drug; further market information is given in chapter 1. Figure IV.1 shows a general overview of the route for synthesis of Amox.

Biofunctionalization and self-interaction chromatography in PDMS microchannels

Abstract A dynamic model is developed that simulates a large size, full scale animal bone demineralization process, which is operated as a simulated moving bed system. The model at the particle level, is based on the unreacted shrinking core model. To solve the system of couples PDEs and ODEs the simulating package gPROMS is used. The model described is succesfully applied to find the optimal process conditions and to increase the productivity of the existing significantly.