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.

Selection of pH-related parameters in ion-exchange chromatography using pH-gradient operations.

This work demonstrates that the type of ion-exchanger (anion or cation), the mode of operation (bind-and-elute or flow-through), and the operational pH of ion-exchange chromatography (IEX) can be selected in a fast and rational way by analytical pH-gradient IEX operations, thereby eliminating the need for pH scouting or high-throughput screening. The developed approach was applied for the selection of an IEX process for the capture of a monoclonal antibody (MAb) from hybridoma cell culture supernatant (CCS). It was found within a day that MAb can optimally be captured by bind-and-elute mode cation-exchange chromatography (CEX) at pH 4.

Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths.

Question: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties?

Approach: Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT-heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 microM of that complex.

Results: The Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e.

pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations.

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9.

Efficient solubilization of inclusion bodies.

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer.

Phase behavior of an intact monoclonal antibody.

Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize.

Design of self-interaction chromatography as an analytical tool for predicting protein phase behavior.

Solution conditions under which proteins have a tendency to crystallize correspond to a slightly negative osmotic second virial coefficient (B22). A positive B22 value guarantees no crystallization to occur. On the other hand, a B22 value within the so called "crystallization slot" thermodynamically supports the crystallization processes but does not guarantee successful crystal growth.

Quantitative analysis in nanoliter wells by prefilling of wells using electrospray deposition followed by sample introduction with a coverslip method.

In contrast to performing assays on a substrate using immobilization techniques, wet analysis in nanoliter-sized wells allows quantitative monitoring of enzyme-based reactions by measuring luminescence with time. However, a suitable dispensing method is required to accurately deposit stabilized enzyme solutions into nanoliter wells in such a manner that the enzyme activities are preserved prior to and during measurements. Furthermore, an efficient method is required to add sample liquid to these wells in such a manner that evaporation of assay liquid is completely prevented during sample introduction and monitoring.

Nanoarrays: a method for performing enzymatic assays.

Conventional enzymatic assays for alcohol dehydrogenase, pyruvate kinase, and enolase performed in 96-well microtiter plates were compared with assays monitored in 25-well nanoarrays. All miniaturized reactions could be performed in maximum volumes of 6.3-8 nL and were read out with a conventional fluorescence microscope system equipped with a scientific grade CCD camera.

Chemical and physical processes for integrated temperature control in microfluidic devices.

Microfluidic devices are a promising new tool for studying and optimizing (bio)chemical reactions and analyses. Many (bio)chemical reactions require accurate temperature control, such as for example thermocycling for PCR. Here, a new integrated temperature control system for microfluidic devices is presented, using chemical and physical processes to locally regulate temperature.

A coverslip method for controlled parallel sample introduction into arrays of (sub)nanoliter wells for quantitative analysis.

We have developed a straightforward coverslip method that combines rapid sample introduction into arrays of 19.8-55.0-microm-deep microwells (prefilled with reactants) on a chip with complete sealing of these wells to avoid evaporation during optical detection.

Miniaturized analytical assays in biotechnology.

Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques. In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.

On-chip contactless four-electrode conductivity detection for capillary electrophoresis devices.

In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-microm-wide, and 20-microm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection.

Considerations on contactless conductivity detection in capillary electrophoresis.

Nearly all analyses by capillary electrophoresis (CE) are performed using optical detection, utilizing either absorbance or (laser-induced) fluorescence. Though adequate for many analytical problems, in a large number of cases, e.g.

Rapid sampling for analysis of in vivo kinetics using the BioScope: a system for continuous-pulse experiments.

In this article we present a novel device, the BioScope, which allows elucidation of in vivo kinetics of microbial metabolism via perturbation experiments. The perturbations are carried out according to the continuous-flow method. The BioScope consists of oxygen permeable silicon tubing, connected to the fermentor, through which the broth flows at constant velocity.

Novel approach for fritless capillary electrochromatography.

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters.

Use of bioaffinity interactions in electrokinetically controlled assays on microfabricated devices.

In this contribution, the role of bioaffinity interactions on electrokinetically controlled microfabricated devices is reviewed. Interesting applications reported in the literature include enzymatic assays, where enzyme and enzyme inhibition kinetics were studied, often in combination with electrophoretic separation. Attention is paid towards developments that could lead to implementation of electrokinetically controlled microdevices in high-throughput screening.