Glycosylation is a critical characteristic of biotherapeutics because of its central role in in vivo efficacy. Multiple factors including medium composition and process conditions impact protein glycosylation and characterizing cellular response to these changes is essential to understand the underlying relationships. Current practice typically involves glycosylation characterization at the end of a fed-batch culture, which in addition to being an aggregate of the process, reflects a bias towards the end of the culture where a majority of the product is made. In an attempt to rigorously characterize the entire time-course of a fed-batch culture, a real-time glycosylation monitoring (RT-GM) framework was developed. It involves using the micro sequential injection (μSI) system as a sample preparation platform coupled with an ultra-performance liquid chromatography (UPLC) system for real-time monitoring of the antibody glycan profile. Automated sampling and sample preparations were performed using the μSI system and this framework was used to study manganese (Mn)-induced glycosylation changes over the course of a fed-batch culture. As expected, Mn-supplemented cultures exhibited higher galactosylation levels compared to control while the fucosylation and mannosylation were consistent for both supplemented and control cultures. Overall, the approach presented in the study allows real time monitoring of glycosylation changes and this information can be rapidly translated into process control and/or process optimization decisions to accelerate process development.

Microsequential injection (microSI) has been successfully coupled with capillary electrophoresis (CE). Presented is the microSI-CE system, interfaced with an integrated Lab-on-Valve (LOV) manifold that provides an efficient sample delivery conduit and a versatile means of sample pretreatment along with total automation of the separation process. Programmable microSI protocols control all critical system peripherals to perform various types of CE sample injections automatically such as electrokinetic (EK) injection, hydrodynamic (HD) injection, and head column field amplification (HCFA) sample stacking injection. Novel features of the microSI-CE technique are demonstrated on assays of samples containing 10 anions that had been used previously as a model system. Calibration studies by EK sample injection yielded linear concentration ranges of 0.5-3.0 mM with linear regression responses of r2 = 0.9999 for both chloride and sulfate using conductivity corrected peak area (CCPA) as concentration responses. Calibration using an internal standard was studied at the same concentration range giving r2 = 0.9992 for both chloride and sulfate and r2 = 0.9997 for both when CCPA correction was deployed. With HCFA sample stacking injection, a linear concentration dynamic range of 0.034-3.419 mM for chloride and 0.014-1.408 mM for sulfate were produced with linear regression responses of r2 = 0.9999 for chloride and r2 = 0.9998 for sulfate.

A "Lab-on-Valve" manifold operated in the micro sequential injection (microSI) mode was adopted to accommodate EPA-approved methods for spectrophotometric determinations of nitrate, nitrite and orthophosphate in the ppb (N or P) concentration range. A computer programmable microSI protocol, utilizing stopped-flow within a copperized Cd-foil filled microcolumn was developed for nitrate reduction to nitrite with subsequent colorimetric measurement, yielding concentration ranges for nitrate of 100.0-4000.0 ppb (N) and for nitrite of 30.0-4000.0 ppb (N) and linear calibration responses of r2 = 0.9999 for nitrate and 0.9995 for nitrite. Using a stopped-flow reaction rate measurement, phosphate was determined in the range 1.0-30.0 ppb (P) with a calibration response of r2 = 0.9997. The technical improvement of this methodology, apart from micro miniaturization, is the use of the stopped-flow technique, that resulted in improved detection limits and allowed reagent consumption to be reduced 1500-fold compared with conventional procedure while the amount of metallic cadmium was reduced 20-fold compared with the EPA-approved continuous-flow assay.

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.

Using an integrated lab-on-valve manifold in a microfluidic sequential injection format (microSI), automated sample processing has been developed for off-line and on-line monitoring of small-scale fermentations. Spectrophotometric assays of ammonia, glucose, glycerol, and free iron were downscaled to use micro-quantities of commercial reagents. By monitoring the reaction rate, the response curves in a stopped-flow mode generate linear calibration curves for ammonia [r2 = 1.000 (0.9% SE)], glycerol [r2 = 0.999 (1.1% SE)], glucose [r2 = 0.999 (1.1% SE)], and free iron [r2 = 0.999 (1.5% SE)]. Since sample dilution and reagent quantities are easily adjusted within the programmable SI format, the lab-on-valve system can accommodate samples over a wide concentration range (ammonia: 3-1200 ppm; glycerol: 20-120 ppm; glucose: 35-1000 ppm; and free iron: 80-400 ppm). This work demonstrates the key advantages of miniaturization through the reduction of sample and reagent use, minimizing waste and providing a compact yet reliable instrument. The lab-on-valve manifold uses a universal hardware configuration for all analyses, only requiring changes in software protocol and choice of reagents. All of these features are of particular importance to small-scale experimental fermentation where multiple analyte analyses are needed in real-time using small sample volumes. It is hoped that this first real-life application of the lab-on-valve manifold will serve not only as a model system to downscale assays in a practical fashion, but will also inspire and promote the use of the integrated microSI manifold approach for a wider range of biotechnological applications.