Bioapplications of Lab-on-a-chip

Nesredin Mussa, Kee Cheung, Imtiaz Alam and Steve Flatman from Lonza Biologics discuss the use of Agilent Technologies' 2100 bioanalyzer in the biopharmaceutical industry for the assessment of therapeutic proteins' titre, integrity and purity

The number of therapeutic antibodies and recombinant proteins produced has risen dramatically over the past decade.The increase in therapeutic antibody research followed the success of Rituxan and Herceptin, the first therapeutic antibodies accepted by the FDA for non-Hodgkin's lymphoma and breast cancer respectively, and lately immunotherapeutic research has shown indications of the inhibition of prion replication and the ability to slow down disease progression.¹ More than 90% of the potential therapeutic antibodies are in clinical trials.

This trend is likely to increase with the timing of proteomic research. The types of diseases targeted include arthritis,² asthma,³ HIV⁴ and stroke.⁵ Clinical trials and commercialised therapeutic antibodies are produced in vast quantities, depending on market requirements and trial demands.

The process of manufacturing a therapeutic protein involves the continuous monitoring of several key parameters, namely titre, structural integrity and purity at the early development stages. Traditional methods such as Protein A HPLC, SDS PAGE electro-phoresis and ELISA assays are used to obtain this information, but these methods are time-consuming, labour-intensive and individually offer only single parameter analysis required for the product quality assessment. However, current advances in proteomic research and increased therapeutic antibody development require more efficient technologies and platforms. These need to have multi-analytical/parameter capabilities to deliver the data rapidly with minimum resources. This is where Agilent's 2100 bioanalyzer (figure 1) proves its usefulness.

Lonza Biologics, part of Swiss-based Lonza Group, is a contract manufacturer of biotherapeutics derived from mammalian cell culture.

At Lonza Biologics the 2100 bioanalyzer has been used during early antibody production to determine product purity, integrity profile, size and titre using minimum sample volume and time resource input for the analysis of monoclonal antibodies (MABs, IgG4) obtained from the Lonza Biologics GS-expression system.

multiple capture

Suitable medium selection was facilitated using 2100 bioanalyzer screening. In-process culture supernatant and purification samples from lab-scale and pilot scale processes were directly tested for product quality and quantity. The data were generated rapidly using the 2100 bioanalyzer and proved useful in the optimisation of the fermentation and purification processes. The 2100 bioanalyzer is part of the new generation of miniaturised µ-TAS (micro total analytical system) allowing the multiple capture of analytical parameters for product quality and cell antibody assembly efficiency. It facilitates microfluidic technology that allows active control of fluids in micro fabricated channels that are only a few micrometers wide.

Microfluidic systems are capable of emulating pumps, valves and dispensers for sample handling on the chip, a separation column for capillary electrophoretic separation, and a reaction system. The technology aims to integrate several sequential experimental steps into one process to obtain a complete laboratory on a chip.

Method and sample preparation was as follows: 4µl of sample was added to 2µl of sample buffer (± ß-MCE) and the sample was heated in boiling water for 1 minute. After cooling, the sample was spun at 13,000 RPM for 15 seconds. This was followed by the addition of 84µl of distilled water. The chip was prepared for the experiment by filling three wells with 12µl acrylamide gel-dye mixture and a well was filled with 12µl destaining solution.

Each chip has a capacity of 10 samples and each kit bears 25 chips. Six microlitres of sample and ladder were applied into the protein chip wells where proteins are separated by the acrylamide sieving process. The protein chip was loaded onto the 2100 bioanalyzer and each protein was then induced by fluorescent excitation at 633nm. All the reagents are provided in the 2100 Bioanalyzer-protein 200 plus kit.6 Biosizing software was used for this study.

Data analysis is automatically performed in real-time and stored and archived in digital format. Data for individual constituents of a complex mixture are shown along with calculations of concentration and percentage total for each protein peak in the trace.

At the present time the device is controlled routinely by 2100 expert software that accumulates raw data and allows data evaluation. Agilent supplies software with IQ & OQ/PV tools and services, and a security pack tailoring the instrument for use in regulated environments will be introduced shortly.

Antibody analysis using the 2100 bioanalyzer at early stages of the manufacturing development process showed that titre and antibody integrity information are essential in the selection process of cell line clones.

Rapid trending of the product titre and overall profile of the fermentation medium containing the product in real time at line was realised using the 2100 bioanalyzer and the specific Lonza Biologics sample preparation method. The titre, total % and quality of the product and other product related fragments were trended at line, figure 2.

The product purification processes require on-going monitoring of the antibody after each purification step because this has a direct impact on the purification development strategy. The 2100 bioanalyzer provides titre, % purity and quality of the product data at line, figure 3. The machine was used for the medium selection process. A variety of medium specifications, including protein-free media, were used to grow CHO cells and the intact antibody, light chain (LC) and heavy chain (HC) products were analysed, figure 4. The 2100 bioanalyzer was compared with Protein A HPLC and A280 techniques. The correlation obtained was R2= 0.98 and R2= 0.99 respectively (data not shown).

efficiency impact

The data presented in this paper shows the applicability of 2100 bioanalyzer for the simultaneous determination of the titre, integrity, and % purity of monoclonal antibodies throughout the product manufacturing and purification development process.

Acceptable correlation with conventional techniques has been seen, and Lonza has demonstrated that the analyser has a positive impact on the efficiency of the manufacturing development production processes.

It can be used for the analysis of both monoclonal antibodies and other recombinant proteins produced in mammalian cell culture. The 2100 bioanalyzer allows at line analysis at a rapid pace relative to conventional techniques.

This introduces improved efficiency in the process of therapeutic antibody selection and manufacturing development, which ultimately reduces the time line required to manufacture products for clinical trials and market supply.