Applications

Product Concentration

AssayMAP® cartridges provide true chromatography capabilities in a high-throughput, miniaturized format from sample volumes ranging from 5 to 200 µl through binding and elution while allowing highly precise control of volume and flow rate to quantitative results. Use these cartridges for microliter-scale separation and sample preparation with analytical instruments including plate readers, mass spectrometers and LC/MS systems. “Open chemistry” supports a broad range of applications from bind-and-elute assays, to ELISAs, to enzymatic digestions, to complex, multi-step sample preparation workflows.

AssayMAP cartridges can be filled with virtually any chromatographic resin or other beaded matrix:

A typical application might include highly selective purification, enzymatic digestion and reversed-phase concentration/desalting steps of the resulting peptides in preparation for MS. These different steps can be integrated on the AssayMAP platform into a single automated workflow, enabling multiple analytes from complex samples (such as cell culture supernatant or plasma) to be determined in a single run. AssayMAP workflows may include multiple wet chemical assays, ELISAs and LC/MS analysis, all run on the same samples on the same system.

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Process Impurity Measurements

Conventional microplate ELISAs are widely used to quantify impurities and contaminants in biopharmaceutical process development. ELISA uniquely combines the versatility, selectivity and sensitivity required for determining concentrations of specific impurities (such as leached protein A or insulin) or general host protein (HCP ELISA) to the ppm level in the presence of high product concentrations. However, processing ELISAs manually can take from 4 to 24 hours and results can vary greatly according the user expertise and technique. Increasing demands from Speed-to-Clinic and Quality-by-Design (QbD) initiatives have turned ELISAs into a major process development bottleneck.

Using AssayMAP ELISA cartridges as the solid phase instead of conventional microplates allows simple conversion of these manual assays into highly precise, automated ones that can use currently validated ELISA reagents. Samples and reagents are pumped at controlled flow rate through cartridges packed with non-porous polystyrene beads that have a binding surface area similar to the bottom of a microplate well. Beads offer a diffusion path on the order of tens of microns, significantly shorter than the several millimeters of a microplate, which allows improved mass transport and allows each reaction step to go to completion in just a few minutes.

An automated AssayMAP ELISA offers:

Pumping substrate through the cartridge at a controlled flow rate reacts with bound enzyme to produce product that is collected in a microplate well and read in a standard plate reader. Assay sensitivity can be controlled by either reducing the flow rate to offer a longer enzyme/substrate reaction time and yield a stronger product signal for a given concentration or by increasing the flow rate to enable higher concentration samples to stay within range.

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Product Variant Analysis

Minimizing the presence of product variants is essential for producing a safe, reliable, and predictable product. Tools that help avoid their formation or help remove or qualify them during the purification process are essential, but can be especially difficult when the characteristics of a product variant are very similar to those of the product itself. BioSystem Development is committed to creating tools that allow identification of variants and monitoring the process to track the origin of the variant. Incorporating chemical separation resins that are compatible with the known product variants in your processes with the miniaturized AssayMAP cartridge technology and automation platform offers a means to monitor these variants by collecting small volume samples throughout the process.

Process Design Space

High‐throughput chromatographic method development becomes increasingly important as the bioprocess industry works to meet the challenges posed by the Quality by Design (QbD) initiative. A common approach is parallel batch adsorption experiments in filter microplate format where 5 to 20 μL of a resin slurry is transferred into the wells, incubated with feedstream samples and separated by vacuum or centrifugation. Traditional equilibrium binding, washing and elution steps are performed and assayed for product and impurities. This approach is relatively complex to automate, has multiple wash and elution steps that can lead to poor yield and reproducibility and cannot be used to observe dynamic binding capacity and other non-equilibrium separation effects. The other major approach is to use small columns operated in parallel on a standard robotic liquid handler. Because these columns are typically 5 mm in diameter, bed volumes below 200 μL are not very practical, which means that a significant volume of feedstream is needed to perform some experiments.

With a 5 μL bed volume of AssayMAP cartridges, precise flow rates down to 1 μL per minute or less is required to achieve residence times appropriate for process chromatography. This is accomplished using patent‐pending “probe syringe” technology where pistons in a multi‐channel liquid handling head have been replaced with ultra‐low dead volume syringes equipped with a special probe. Mounting a cartridge to the probe provides a direct fluid connection between the top of the bed and the interior of the syringe through the cartridge inlet seal that enables precise, positive displacement pumping in both directions through the bed. Currently a 12‐ channel workstation is offered; a 96‐channel version is under development.

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