Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein.

Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pKa value(s), and the amount of immobilized protein.Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

Development of an electrically responsive hydrogel for programmable in situ immobilization within a microfluidic device.

A novel microfluidic channel device with programmable in situ formation of a hydrogel 3D network was designed. A biocompatible hybrid material consisting of iron ion-crosslinked alginate was used as the active porous medium. The sol-gel transition of the alginate was controlled by the oxidation state of Fe ions and regulated by an external electrical signal through an integrated gold plate electrode. The SEM images, FT-IR analysis, and rheological test demonstrated that homogeneous yet programmable hydrogel films were formed. The higher the concentration of the crosslinker (Fe(iii)), the smaller the pore and mesh size of the hydrogel. Moreover, the hydrogel thickness and volume were tailored by controlling the deposition time and the strength of electric current density. The as-prepared system was employed as an active medium for immobilization of target molecules, using BSA as a drug-mimicking protein. The reductive potential (activated by switching the current direction) caused dissolution of the hydrogel and consequently the release of BSA and Fe. The diffusion of the entrapped molecules was optimally adjusted by varying the dissolution conditions and the initial formulations. Finally, the altering electrical conditions confirm the programmable nature of the electrically responsive material and highlight its wide-ranging application potential.

Designing scalable ultrafiltration/diafiltration process of monoclonal antibodies via mathematical modeling by coupling mass balances and Poisson-Boltzmann equation.

Ultrafiltration/diafiltration (UF/DF) operations are employed for achieving the desired therapeutic monoclonal antibody (mAb) formulations. Due to electrostatic interactions between the charged proteins, solute ions, and uncharged excipients, the final pH and concentration values are not always equal to those in the DF buffer. At high protein concentrations, typical for industrial formulations, this effect becomes predominant. To account for challenges occurring in industrial environments, a robust mathematical framework enabling the prediction of pH and concentration profiles throughout the UF/DF process is provided. The proposed mechanistic model combines a macroscopic mass balance approach with a molecular approach based on a Poisson-Boltzmann equation dealing with electrostatic interactions and accounting for protein exclusion volume effect. The mathematical model was validated with experimental data of two commercially relevant mAbs obtained from an industrial UF/DF process using scalable laboratory equipment. The robustness and flexibility of the model were tested by using proteins with different isoelectric points and net charges. The latter was determined via a titration curve, enabling realistic protein charge-pH evaluation. In addition, the model was tested for different DF buffer types containing both monovalent and polyvalent ions, with various types of uncharged excipients. The model generality enables its implementation for the UF/DF processes of other protein varieties.

A frontal analysis combined with a simultaneous chromatographic analysis of macromolecules using a single chromatographic system.

A chromatographic system was adapted to allow monitoring of eluent of preparative column via absorbance and with the chromatographic analysis of the target macromolecule on the same chromatographic system. The proposed approach was tested on important macromolecules, such as monoclonal antibodies, monoclonal antibody aggregates and plasmid DNA (pDNA). A frontal analysis was made on the preparative column, while a chromatographic on-line analysis was performed by sequentially injecting the preparative column outlet on a convection-based analytical column, operating on the same chromatographic system. Cation and/or anion exchangers were used as the chromatographic media (along with a protein A), depending on the sample to be purified. The method was found to be robust and reproducible. To adjust the limit of detection, an algorithm varying the number of injections was used, enabling accurate monitoring of an early breakthrough for concentrations below 1% of the feed concentration. The accuracy varies according to the applied flow rate, but it is typically in the range of few percent, or even below. Due to its simplicity and flexibility, the proposed method can be easily adapted to a pharmaceutical environment.