Chips with everything

Lab-on-a-chip technology is rapidly being adopted for QC of bioproducts because of its ease of use and reproducibility, say Tony Owen and Meike Kuschel, of Agilent Technologies

The term 'lab-on-a-chip' is applied to products based on two very different technologies. One group is the planar devices, such as DNA microarrays, which have thousands of individual test sites deposited on a wafer of glass or similar material. These devices are currently used almost exclusively for research in life sciences and have not yet found significant application in manufacturing.

The second and larger group utilises microfluidic technology. These devices manipulate small amounts of fluids in microchannels fabricated in glass or other materials. Although commercial development of products using this technology became available several years after microarray technology they are already finding application in manufacturing and this is expected to increase rapidly in the future.

adopted techniques

Microfluidic lab-on-a-chip devices can be characterised as a having at least one but more usually a matrix of channels with cross section of less than 1mm but typically 20-50µm.

They are fabricated in silicon and glass by photolithography and etching, techniques adapted from the microelectronics sector; indeed lab-on-a-chip seeks to achieve the same goals as the microelectronics industry ; the shrinking of processes, in this case chemical and analytical, to very small dimensions.

Typically a number of functional elements are combined together on a chip to make the lab-on-a-chip. Common elements are:

• Liquid transportation and mixing. The simplest way to move liquid in a chip is pressure but the most commonly used method is electro-kinetic or electro-osmotic driven flow. By the combining of channels in 'crosses', 'Ts' and other configurations and switching the electrical fields in different sequences it is possible to create virtual 'valves' to switch flows along multiple paths or merging with a diluent or reagent.

• Separation. Molecular separation can be performed on the chip using chromatographic or, more commonly, electrophoretic processes. Electrophoresis separates molecules according to their different charges. Where no charge difference is present the use of a 'sieving' polymer in the channels can be used to separate molecules by size.

• Reaction. Sections of the device can be heated electrically to promote chemical or physical processes.

• Detection. Some forms of detection (e.g. electrochemical or simple optical absorbance) can be integrated directly on the chip, but most commonly the chip is designed with an appropriate detection window or cell and the detector (most commonly laser- induced fluorescence) is external to the chip.

By combining these elements the laboratory-on-a-chip is created. Currently the number of elements combined on a chip ; and thus the total functionality ; is limited, but chips are becoming increasingly complex as the technology improves.

Most commercial lab-on-a-chip systems consist of 'chips' made of glass or plastic that are placed on or in an instrumental platform that provides the pressure supplies, power and electronics for control and detection.

The advantages of microfluidic systems include:

• Very small sample requirements

• Low reagent usage

• Fast analysis thanks to the small size

• Small instrumental footprint that takes less bench space or, together with lower power consumption, enables portability

• Improved reliability and reproducibility

• Potentially inexpensive because may use mass production techniques

However, microfluidics is not without its problems. For example, the small dimension means that, relative to macro systems, there is a much higher surface-to-volume ratio and any surface effects are amplified and must be controlled very carefully. Also clogging of channels due to particles or depositions can be an issue because of the small channel dimensions.

The first fully commercialised implementation of microfluidics, and the most successful to date, is the Agilent 2100 bioanalyser, which offers electrophoretic DNA, RNA and protein analysis and, more recently, pressure-driven cell assays. Similar in concept is the Hitachi Cosmo I that uses disposable plastic chips for the analysis of DNA but is currently available only in Japan. The Shimadzu MCE-2010 Microchip Electrophoresis System is essentially a miniaturised capillary electrophoresis system with UV detection and is also used for DNA analysis

functional performance

Cepheid is developing fluidic cartridges to carry out automatically the complex steps of DNA extraction from a variety of sample types. Disposable, single-use cartridges are designed to perform the functions such as reagent containment and delivery, sample and reagent aliquoting and mixing, cell separation and concentration, cell lysis using ultrasonic techniques, DNA or RNA capture, purification, preparing reaction mixture and filling integrated PCR reaction tube. These systems have found extensive use for bioterrorism pathogen detection.

Other companies include Fluidigm, which fabricates microscale pumps and valves directly within flexible rubber chips using a technique called Multi-layer Soft Lithography, and Gyros, which designs its devices into CDs that are spun to create centrifugal forces that move the sample through the channels.

New technologies are typically first applied to solving novel or intractable problems in research and take a long time to move down the value chain into manufacturing. This is because initially they are often difficult to work with, unreliable and the requirements of GLP and GMP slow down their adoption. In the case of lab-on-a-chip technology the interest from manufacturing has, in certain areas, been virtually instantaneous. The reason for this is that lab-on-a-chip can replace gel electrophoresis which has the disadvantages of being slow and difficult to automate and does not produce digital data directly. Most importantly, gel electrophoresis offers very poor day-to-day and inter-lab reproducibility and is therefore inherently very difficult to validate. In addition, for proteins, toxic chemicals must be used.

Lab-on-a-chip systems that are easy to use, are reproducible, do not use toxic chemicals and produce digital data directly are a huge improvement for the manufacturing environment. The technology has already been adopted by many manufacturers of bioreagents and will be adopted by pharmaceutical companies for stability or product QC as soon as product compliance becomes available.

Stability tests under stress conditions are commonly performed to identify degradation or aggregation products and to evaluate which specific test parameters are the best indicators of product stability and need to be monitored under proposed storage conditions. The limits of acceptable degradation are derived from the analytical batch profiles of drug substances used in the preclinical and clinical studies.

The degree of purity or the amount of degradation products needs to be reported and documented. The Agilent 2100 bioanalyser enables automatic determination of the amount of degradation products in antibody batches (under reducing conditions or non-reducing conditions on the same chip) within 45 minutes for 10 samples.

Furthermore, the analysis is highly reproducible with a relative standard deviation of 2% (figure 2). The standard samples can be easily compared to the stability samples by using the overlay feature of the software.

separation failure

A portion of IgG4 immunoglobulins is secreted as half-antibodies or half-molecules when inter-heavy chain disulfide bonds are absent. The amount of half-molecules is an important QC criterion for IgG4 manufacturing. Half-molecules are detectable by traditional non-reducing SDS-PAGE.

Other non-denaturing methods, such as native PAGE and gel filtration HPLC, fail to separate half-molecules from the intact antibody. Lab-on-a-chip analysis allows the determination of half-molecule content in IgG4 preparations with accuracy and precision comparable to non-reducing SDS PAGE.

In addition, this method offers the advantage of automation, easier sample handling, faster turnround times and streamlined analysis of the electronic data presented in a chromatogram-like fashion.