Balancing the Affinity and Pharmacokinetics of Antibodies by Modulating the Size of Charge Patches on Complementarity-Determining Regions.

A localized positive charge on IgG (referred to as a "charge patch") shows an adverse effect on pharmacokinetics (PK), so it would seem to be best practice to avoid charge patches during the discovery stage and closely monitor charge interactions during the development process. In certain circumstances, however, charge patches are required for target binding, in which case completely removing charge patches is not feasible. Therefore, quantitative measurement of a charge patch and its impact on PK is critical to the success of therapeutic antibody development. In this article, we generated mutations of a recombinant human antibody (referred to as mAb1) with disrupted charge patches to investigate how charge patches on IgG antibodies impact both target-binding affinity and PK-related factors. We conclude that it is important to modulate the size of the charge patch in order to balance target-binding affinity and PK.

Mini-scale bioprocessing systems for highly parallel animal cell cultures.

Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.

An actively mixed mini-bioreactor for protein production from suspended animal cells.

Biopharmaceutical production would benefit from rapid methods to optimize production of therapeutic proteins by screening host cell line/vector combination, culture media, and operational parameters such as timing of induction. Miniaturized bioreactors are an emerging research area aiming at improving the development speed. In this work, a 3 mm thick mini-bioreactor including two 12 mm wide culture chambers connected by a 5 mm wide channel is described. Active mixing is achieved by pressure shuttling between the two chambers. Gas-liquid phase exchange for oxygen and carbon dioxide is realized by molecular diffusion through 50 microm thick polymethylpentene membranes. With this unique design, a velocity difference between the middle area and the side areas at the interfaces of the culture chambers and the connecting channel is created, which enhances the mixing efficiency. The observed mixing time is on the order of 100 s. The combination of high permeability toward oxygen of polymethylpentene membranes and fluid movement during active pressure shuttling enables higher volumetric oxygen transfer coefficients, 5.7 +/- 0.4-14.8 +/- 0.6 h(-1), to be obtained in the mini-bioreactors than the values found in traditional 50 mL spinner flasks, 2.0-2.5 h(-1). Meanwhile, the calculated volume averaged shear stress, in the range of 10(-2)-10(-1) N/m(2), is within the typical tolerable range of animal cells. To demonstrate the applicability of this mini-bioreactor to culture suspended animal cells, the insect cell, Spodoptera frugiperda, is cultured in mini-bioreactors operated under a K(L)a value of 14.8 +/- 0.6 h(-1) and compared to the same cells cultured in 50 mL spinner flasks operated under a K(L)a value of 2.2 h(-1). Sf-21 cells cultured in the mini-bioreactors present comparable length of lag phases and growth rates to their counterparts cultured in 50 mL spinner flasks, but achieve a higher maximum cell density of 5.3 +/- 0.9 x 10(6) cell/mL than the value of 3.4 +/- 0.4 x 10(6) cell/mL obtained by cells cultured in 50 mL spinner flasks. Sf-21 cells infected with SEAP-baculovirus produce a maximum SEAP concentration of 11.3 +/- 0.7 U/mL when cultured in the mini-bioreactor. In contrast, infected Sf-21 cells cultured in 50 mL spinner flasks produce a maximum SEAP concentration of 7.4 +/- 0.9 U/mL and onset of production is delayed from 18 h in minibioreactor to 40 h in spinner flasks.

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis.

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

A surface modification strategy on silicon nitride for developing biosensors.

A surface modification strategy for the use of giant magnetoresistive materials in the detection of protein-protein interactions is developed. This modification strategy is based on silanization of semiconductive materials. A native silicon nitride surface was treated with concentrated hydrofluoric acid to improve surface homogeneity. Nano-strip was used to oxidize silicon nitride to form a hydrophilic layer. Aminopropyltriethoxysilane was subsequently used to functionalize the treated surfaces to form amine groups, which were further activated with glutaraldehyde to introduce a layer of aldehyde groups. The effectiveness of this modification strategy was validated by chemiluminescence immunoassays of purified 6x His-HrpW of Pseudomonas syringae pv. tomato DC3000 and human transferrin. Signals with intensities related to concentrations of these two immobilized model proteins were observed. The modified surface was also validated by a more complex system: intercellular proteins secreted by DC3000. HrpW in these protein mixtures was successfully recognized by anti-HrpW antibodies when mixed proteins were immobilized onto activated surfaces. This surface modification strategy provides a platform onto which proteins can be directly immobilized for biosensor and protein array applications.