Miniaturised and automated protein quantification for more efficient development of biotherapeutics

Gyrolab Bioaffy® CD microlaboratories miniaturize and automate protein quantification, delivering the highest level of accuracy and reproducibility. Scientists get results similar to ELISA, only more quickly, more easily and at lower cost.

Open, flexible platform

A Gyrolab® microlaboratory miniaturizes and integrates the many steps of conventional laboratory processes without the need for mechanical functions such as valves and pumps. Application-specific microlaboratories use a modular design offering the potential to run different applications on a single instrument and to create customized solutions.

Gyros technology is well-suited for many growing areas within the life science arena, healthcare and diagnostic markets. One of the company’s main focus areas is the biopharmaceutical industry and especially applications related to protein quantification within the development of therapeutic monoclonal antibodies.

Protein quantification using Gyrolab Bioaffy

Gyrolab Bioaffy uses the CD technology in the format of a flow-through immunoassay, with CDs being processed in a software controlled and fully automated instrument.

The open design principle facilitates quantification of any target protein for which an immunoassay can be developed, and allows experimental set-up to be designed on a day-to-day basis. Each CD contains individual microstructures in which samples are analyzed in parallel. The CD microstructures are assembled in segments, each segment containing 8 microstructures connected by a common channel. Each microstructure contains a column (15 nL) pre-packed with streptavidin-coated beads. Capture reagents (commercially available reagents, biotinylated using NHS-sulfo-LC-biotin, Pierce) are then bound to the beads creating protein-specific columns.

Sandwich immunoassays miniaturized and integrated into a CD microlaboratory.

Gyrolab Bioaffy has the flexibility to meet diverse analytical requirements. In general, assays provide a measuring range of up to 104, and with over 100 data points processed in less than an hour Gyrolab Bioaffy is capable of significantly improving throughput.

Fully automated analysis

CDs are run in Gyrolab® Workstation LIF, a fully automated system where samples and reagents are transferred sequentially to specific inlets on the CD by a robotic arm. Liquid enters the microstructures by capillary action and the required volumes are defined within each microstructure. Samples and reagents are moved through the microstructure by spinning the CD at pre-programmed intervals and speeds to create the desired flow rates. Detection begins as soon as the reactions are completed using a laser-induced fluorescence detector integrated in the workstation. Fluorescence is detected by rotating the CD while the detector moves along the radial direction of the CD. Thus, an image of the localized fluorescence intensity of each individual column is created. The total integrated fluorescence of each column corresponds to the total protein bound to the column.

Case study:

Quantification of monoclonal IgG and host cell proteins during biopharmaceutical process development

In collaboration with a major pharmaceutical company, Gyrolab Bioaffy and Gyrolab Workstation LIF were successfully used for quantification of monoclonal IgG and host cell proteins (HCP). In addition to the inherent speed and ease of use provided by the system, what made this work particularly interesting to the customer was that the Gyros solution provided a measurement range from micrograms to grams per liter. This allowed dilution-free analysis in concentration ranges relevant for the entire purification process, from cell supernatant to purified product – all on a single system.

Quantifying drug product

In order to generate cost effective processes for manufacturing of monoclonal IgG significant efforts are made to optimize cell culturing conditions and purification procedures in order to increase product consistency and overall product yields. Fast and accurate quantification of the produced material is an essential part of this process.

Current technologies, such as ELISA and HPLC, are able to deliver good quality results but have limitations in terms of throughput and measurement range. As the purification process proceeds from crude cell supernatant to final product, the IgG concentration increases significantly, and ELISA, in particular, increasingly requires pre-assay dilution, which is time-consuming, tedious and can introduce errors.

Quantifying contaminants

A second important factor in the development of biopharmaceuticals is the quantification of protein contaminants originating from cell culturing and fermentation, which can be co-purified along with the product and potentially cause an allergic reaction when the drug is administered to patients. In crude cell supernatants and at the early stages of the purification process high concentrations of contaminating host cell proteins can be present. Concentrations decrease as the purification process progresses, with the aim of minimizing impurities in the final product.

ELISA is one of the most commonly used technologies for the detection of contaminants due to its relatively high sensitivity. However, due to its narrow measurement range, ELISA requires extensive dilution when monitoring contaminants during the early stages of the purification process.

Single system solution

Gyros was able to overcome the drawbacks of the alternative technologies by offering a complete solution with a measurement range from micrograms to grams per liter, thereby, meeting the needs of both applications. The fully automated instrument was used to process different CD microlaboratories: Gyrolab Bioaffy 20 HC for dilution-free quantification in the mg/mL range; and Gyrolab Bioaffy 200 for high sensitivity assays when detecting low concentrations.

The increased capacity in Gyrolab Bioaffy 20 HC is accomplished by equipping the microstructures with columns of high-capacity streptavidin (SA) beads and through reduction of the sample volume used for quantification from 200 nL in Gyrolab Bioaffy 200 to 20 nL in Gyrolab Bioaffy 20 HC.

Quantification of monoclonal IgG

The combination of Gyrolab Bioaffy 20 HC and the selected reagents resulted in a measurement range covering three orders of magnitude, allowing for quantification of monoclonal IgG in the g/L range. Cell supernatant samples were analyzed with regards to content of monoclonal IgG using Gyrolab Bioaffy 20 HC and affinity HPLC absorbance measurements at 280 nm. The two methods showed excellent correlation across a range of concentrations.

In order to investigate the parallelism of the assay for IgG quantification, serial dilutions of four cell culture supernatant samples were analyzed and plotted together with a dilution of the standard preparation. The slopes of the resulting curves all proved to be very similar.

The specificity of the selected reagents to the different subclasses of IgG, and to IgM was determined on Gyrolab Bioaffy 20 HC. The immunoassay was shown to be specific for IgG1, IgG2 and IgG4. As expected, there was no significant binding to IgG3 or IgM.

Gyrolab Bioaffy 20 HC was used to show the specificity of the assay for IgG1, IgG2 and IgG4.

Quantification of host cell proteins

When quantifying host cell proteins, CDs and reagents can be chosen to match the required measurement range. Gyrolab Bioaffy 200 can be used to achieve a high-sensitivity HCP assay with a measurement range covering three orders of magnitude and a limit of detection of approximately 1 ng/mL. If the same assay is performed using Gyrolab Bioaffy 20 HC the measurement range is shifted to the right, allowing, for example, quantification of HCP in crude cell supernatants.

Standard curves for HCP on Gyrolab Bioaffy 200 (red curve) and Gyrolab Bioaffy 20 HC (orange curve).

Monitoring cell culture development

To monitor the development of cell production over time, regarding production of the intended IgG molecule versus production of contaminating proteins, a series of samples collected during the cell cultivation process were analyzed on Gyrolab Bioaffy 20 HC. As the cultivation progresses the concentration of produced IgG increases. The various increases in concentration of IgG were accurately measured for six different clones between day eight and day twelve of cultivation. The corresponding analysis of HCP concentration was also measured. Samples were again collected after eight and twelve days of cultivation and as expected there was a significant increase in HCP concentration over time.

Conclusions from the study

Gyrolab CD microlaboratories with Gyrolab Workstation LIF were successfully used for quantification of monoclonal IgG and HCP in concentration ranges relevant for the entire purification process from cell supernatant to purified product. Assay steps were integrated and miniaturized in the CD format, increasing throughput and simplifying working methods, whilst still delivering reliable, high quality results.

Gyros provides a fully automated, flexible solution for maximizing the efficiency of biopharmaceutical process development. The open platform makes it possible to adjust assay conditions, such as CD type and reagents, to match analytical requirements.

For more information about Gyros and its technology, visit www.gyros.com.