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Biomedical subjects

Kaushal Rege

Publications and source records attributed to Kaushal Rege.

8 recordsLinked to original sources
PubMed2006

High-throughput process development for recombinant protein purification.

Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel screening of a wide variety of actual bioprocess media and conditions and represents a novel paradigm approach for the high-throughput process development of recombinant proteins.

Anion Exchange Resins↗
PubMed2005

Multidimensional high-throughput screening of displacers.

A multidimensional, batch high-throughput screening (MD-HTS) protocol was developed to investigate the effects of various parameters on the selectivity of ion-exchange protein displacement systems. A variety of molecules were screened, and the results were employed to provided insights into the influence of displacer chemistry and concentration, resin chemistry, and mobile-phase salt counterion on the efficacy and selectivity of these nonlinear chromatographic systems. These results open up the possibility of tailoring the selectivity of displacement separations by choosing appropriate combinations of operating conditions using the MD-HTS technique. The screens were also employed for the identification of displacers and conditions for the separation of a challenging protein mixture by selective displacement chromatography. Column displacements were carried out with potential lead compounds identified from the MD-HTS screens, and the results confirmed that selective displacement could indeed be achieved for this model mixture. Furthermore, the results indicated that this approach is particularly useful when the order of elution is not changed, but the inherent selectivity is increased in the presence of the displacer. The results presented in this paper demonstrate the utility of the MD-HTS technique for rapid method development in protein ion-exchange displacement chromatography.

Calcium↗
PubMed2005

A priori prediction of adsorption isotherm parameters and chromatographic behavior in ion-exchange systems.

The a priori prediction of protein adsorption behavior has been a long-standing goal in several fields. In the present work, property-modeling techniques have been used for the prediction of protein adsorption thermodynamics in ion-exchange systems directly from crystal structure. Quantitative structure-property relationship models of protein isotherm parameters and Gibbs free energy changes in ion-exchange systems were generated by using a support vector machine regression technique. The predictive ability of the models was demonstrated for two test-set proteins not included in the model training set. Molecular descriptors selected during model generation were examined to gain insights into the important physicochemical factors influencing stoichiometry, equilibrium, steric effects, and binding affinity in protein ion-exchange systems. The a priori prediction of protein isotherm parameters can have direct implications for various ion-exchange processes. As proof of concept, a multiscale modeling approach was used for predicting the chromatographic separation of a test set of proteins using the isotherm parameters obtained from the quantitative structure-property relationship models. The simulated column separation showed good agreement with the experimental data. The ability to predict chromatographic behavior of proteins directly from their crystal structures may have significant implications for a range of biotechnology processes.

Adsorption↗
PubMed2004

Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: identification of high-affinity displacers and DNA-binding ligands.

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.

Aminoglycosides↗
PubMed2004

Parallel screening of selective and high-affinity displacers for proteins in ion-exchange systems.

This paper employs a parallel batch screening technique for the identification of both selective and high-affinity displacers for a model binary mixture of proteins in a cation-exchange system. A variety of molecules were screened as possible displacers for the proteins ribonuclease A (RNAseA) and alpha-chymotrypsinogen A (alpha-chyA) on high performance Sepharose SP. The batch screening data for each protein was used to select leads for selective and high-affinity displacers and column experiments were carried out to evaluate the performance of the selected leads. The data from the batch displacements was also employed to generate quantitative structure-efficacy relationship (QSER) models based on a support vector machine regression approach. The resulting models had high correlation coefficients and were able to predict the behaviour of molecules not included in the training set. The descriptors selected in the QSER models for both proteins were examined to provide insights into factors influencing displacer selectivity in ion-exchange systems. The results presented in this paper demonstrate that this parallel batch screening-QSER approach can be employed for the identification of selective and high-affinity displacers for protein mixtures.

Cation Exchange Resins↗
PubMed2002

High-throughput screening and quantitative structure-efficacy relationship models of potential displacer molecules for ion-exchange systems.

A technique is presented for the high-throughput screening of ion-exchange displacers. Potential displacers were employed to displace proteins in parallel batch ion-exchange experiments. The percentage of protein displaced from a particular stationary phase was then used as a parameter to rank the displacers. By employing this technique, a large number of molecules possessing a range of affinities and properties could be rapidly evaluated. This data was then used together with traditional and electron density-based transferable atom equivalent (TAE) molecular descriptors computed for the displacer molecules to produce quantitative structure-efficacy relationship (QSER) models using a genetic algorithm/partial least squares (GA/PLS) regression approach. The QSER models were generated using a portion of the protein-displacement data, with the remainder serving as a test set. Descriptor selection and model building was accomplished using a genetic algorithm/partial least squares approach. The resulting models were found to have high-correlation coefficients and could be used to accurately predict the behavior of molecules not included in the training set. In addition, the models were employed to examine a virtual library of displacers based on modifications of neomycin to provide further insight into displacer design. The results presented here indicate that it may be possible to design displacers that can dramatically improve the effective selectivity of ion-exchange chromatographic materials.

Algorithms↗
PubMedDate not supplied

Investigation of DNA-binding properties of an aminoglycoside-polyamine library using quantitative structure-activity relationship (QSAR) models.

We have recently developed a novel multivalent cationic library based on the derivatization of aminoglycosides by linear polyamines. In the current study, we describe the DNA-binding activity of this library. Screening results indicated that several candidates from the library showed high DNA-binding activities with some approaching those of cationic polymers. Quantitative Structure-Activity Relationship (QSAR) models of the screening data were employed to investigate the physicochemical effects governing polyamine-DNA binding. The utility of these models for the a priori prediction of polyamine-DNA-binding affinity was also demonstrated. Molecular descriptors selected in the QSAR modeling indicated that molecular size, basicity, methylene group spacing between amine centers, and hydrogen-bond donor groups of the polyamine ligands were important contributors to their DNA-binding efficacy. The research described in this paper has led to the development of new multivalent ligands with high DNA-binding activity and improved our understanding of structure-activity relationships involved in polyamine-DNA binding. These results have implications for the discovery of novel polyamine ligands for nonviral gene delivery, plasmid DNA purification, and anticancer therapeutics.

Aminoglycosides↗