Mats Ingañas

Efficiency is more than just a buzzword in today's pharmaceutical industry; declining productivity and diminishing returns on investment have made it an over-arching mindset that is critical to corporate survival. Low-tech strategies to maximize efficiency may include streamlining existing protocols and relieving bottlenecks, but, undoubtedly, the most direct and effective approach to improving process efficiency and overall productivity is to operate 'lean and mean' — to maximize time, labour and resource utilization while simultaneously optimizing the speed and quality of data acquisition — ultimately moving projects through the pipeline more quickly and with greater success. Employing miniaturization and automation solutions to replace processes that consume substantial quantities of high-value resources and rely on labour-intensive manual operations will not only improve the efficiency of critical steps along the drug discovery continuum, but also help increase overall productivity by ensuring robust and reproducible operations. 

Miniaturization and automation technologies have particular advantages and applicability for assay development and high-throughput assay performance. There are many benefits to being able to perform automated assays at the nanoliter scale; for example, smaller volumes mean less sample is needed per assay, allowing companies to perform more assays and gain more information from limited starting materials. This also reduces the amount of reagents and buffers required, translating to smaller quantities of materials that companies have to purchase, dispose of and store. From an efficiency perspective, the most important aspects of assay miniaturization are the reduced time for assay development and the shorter cycle times, enabling more timely decision-making and helping companies move projects forward more rapidly (Figure 1). 

Figure 1: Correlation between Biacore and Gyrolab results in lgG-containing harvest.			

Adding automation and robotics technology to miniaturized platforms further enhances the speed of processing and also introduces other advantages. Automation eliminates the variation and risk of error associated with manual operations, thereby improving data quality (Figure 2). Importantly, by standardizing operations and procedures, automation facilitates the transfer of processes both within a drug discovery organization and between a company and its outsourcing partners. Validated assays performed on readily available instrumentation are particularly attractive to the rapidly expanding CRO and CMO industry. Not only do contract service providers place a premium on cost-effectiveness, efficiency and productivity, they also prefer technology solutions that minimize manpower needs. 

Figure 2: High intra-run reproducibility.			

Biologicals, including protein-based therapeutics and, in particular, monoclonal antibodies, are enjoying a resurgence of interest in the pharmaceutical industry, and this trend is likely to continue. Companies are under intense pressure to identify promising protein therapeutics quickly, to fail unsafe or unsuitable drug candidates early on in the process, and to generate as much information as possible regarding safety and efficacy during the discovery and preclinical stages of product development before taking a drug candidate into the clinical phase. Coupled with these pressures is an industry-wide emphasis on cutting costs, preserving limited resources and minimizing the numbers of animals required for preclinical testing. To meet this last goal, efforts have focused on the development of innovative, high-throughput assays that generate data that can predict a compound's metabolic, toxic and pharmacokinetic (PK) properties. 

Traditionally, immunoassays developed for evaluating protein-based therapeutics on automated platforms relied on the ELISA format and required several weeks, or even months, for assay design and optimization. A severe drawback of ELISA is its relatively narrow dynamic range, which may force users to analyse several sample dilutions in parallel to ensure that useful data will be generated. This introduces variability and inefficiency into the process. 

Gyrolab (Gyros, Sweden), a microfluidics-based automated system, replaces ELISA-based sandwich assays with miniaturized fluorescence-based immunoassays performed on affinity capture columns embedded in microstructures in a compact disc (CD) microlaboratory. By accommodating a selection of CDs together with intuitive software and sensitive visualization tools, the system simplifies assay development and optimization, and performs high-throughput immunoassays in a CD format (Figure 3). Running even a few samples through an ELISA takes at least a full day's work, but results are available on Gyrolab within 1–1.5 h. Furthermore, this miniaturized format may reduce the total cost per sample by up to 50%, including savings on manpower. 

The technology enables quantitative analysis of protein drugs and overcomes many of the problems associated with ELISA, including those related to dynamic range, reaction times and matrix effects. Gyrolab's flow-through assay format offers particular advantages for performing PK assays, with outcomes, cost savings, and time factors far superior to what can be achieved using conventional assay methods. Other applications that can benefit from the speed and accuracy of the technology include antidrug antibody (ADA) assays, detection of bioprocess contaminants and biomarker assays, as well as assays for the characterization of binding properties. An additional benefit is the ease of transitioning ELISA-based immunoassays to Gyrolab. Similarly, the transfer of assays between a pharmaceutical company and its CRO or CMO partners is also straightforward because the Gyrolab protocol for running the assay is automated and has fewer variables compared with ELISA. 

The ability to perform multiplexing yields benefits that include reduced sample consumption and the faster generation of more information with fewer runs. Most immunoassay methods achieve multiplexing by combining multiple reagents and labels together with the sample in a complex mixture, and then detecting and sorting out the various signals after the binding reactions have completed, but this approach complicates assay design and optimization. Multiplexing on Gyrolab involves performing different assays with the same sample or running the same assay using different samples, all on the same CD, in such a way that scarce samples are used in an optimal manner. In this way, multiplexing does not compromise assay optimization or sample preparation. Each assay is individually optimized and then run side-by-side, allowing individual sample preparation for each assay if required. This produces better results just as quickly, but uses smaller sample volumes compared with traditional multiplexing. 

The flow-through principles underlying the CD-based microlaboratory ensure limited contact time between the immobilized solid phase and the sample, permitting desired reactions to occur at a rapid pace, while preventing problematic matrix effects. These can occur during prolonged contact, as is characteristic of the static, microplate-based ELISA format, causing nonspecific binding reactions that impair the performance of the assay.  

Two features that contribute to the Gyrolab's broad dynamic range are the high capacity of the solid phase for the biotinylated capture antibody and the use of fluorescently-labelled antibodies to detect binding of the target protein. Whereas ELISA will seldom yield a dynamic range greater than orders of magnitude, for immunoassays performed on Gyrolab, three to four orders of magnitude is the norm. As a result, the need for repeat testing, and estimating concentration ranges and dilution requirements is reduced. The dynamic range of an assay can be extended even further using a combination of CD types; for example, those that are suitable for high-concentration or high-sensitivity assays. 

A broad dynamic range is of particular benefit for PK studies, as drug molecule concentrations can be very high. Furthermore, assays performed at the nanoliter scale enable users to extract more information from minute sample volumes, allowing repetitive sampling from individual animals without jeopardizing homeostasis. This sequential sampling strategy yields a more complete PK profile (Figure 4). 

Figure 4: Target quantification: changing CD decreases limit of detection.			

By enabling a sequential assay format, the system can eliminate, or at least minimize, the phenomenon known as 'the hook effect'. The high-dose hook effect occurs when the concentration of analyte exceeds the amount of antibody in the assay. Because of the high capacity of the solid phase in the microstructure and the large amount of material it can bind, in combination with efficient washing procedures, assays performed on the CD can accommodate a broad range of sample concentrations and the risk of working in the hook zone is essentially eliminated. 

In a real-world comparison of the CD format and ELISA for the quantification of a monoclonal antibody drug, several dramatic comparisons emerge (Figure 5). For Gyrolab, compared with ELISA, respectively: 

Figure 5: Head-to-head comparison with ELISA for pharmacokinetic study in 20% human serum.									

Measurement range was 13–2500 ng/mL compared with 63–315 ng/mL.

Assay processing times, including sample preparation, were 3 h 

Precision was improved, with a coefficient of variation (CV) of <12% with the flow-through immunoassay, in contrast to <25% CV with ELISA.

Another assay application that could benefit from a microfluidics-based automated solution is immunogenicity testing to detect ADAs. Because ADAs used as positive controls are difficult and costly to produce, the small sample and reagent volumes needed in a miniaturized assay format help minimize resource consumption. Automation of the process allows for high-throughput ADA detection. Selection of the optimal assay format for ADA detection will depend on a number of parameters, such as half-life of the drug, reagent availability and sample pretreatment.  

Immunoassays also play a pivotal role throughout drug development in characterizing the binding properties of candidate drug molecules early on in the discovery process, and then monitoring those properties during further development in terms of affinity or EC50 for the target. Gyrolab provides several approaches for generating this type of information. 

Another valuable application of this technology is highly relevant to protein production and scale-up. The miniaturized immunoassay format can be used to generate analytical data during bioprocess development, detecting and quantitating impurities and host-cell proteins, and measuring product titers. By improving the turnaround time for product analysis and process-related impurity testing, this assay platform enables accelerated decision-making (Figure 6). 

Figure 6: Broad dynamic range for quantification of process-related impurities.			

The descriptions, examples and applications presented here define a valuable role for an automated and miniaturized immunoassay platform. 

With interest in protein-based therapeutics on the rise and a growing need for robust, reproducible, and cost-effective analytical tools and techniques, this technology is being embraced by both pharma and biotech for its ease of use and ability to generate high-quality results. Above all else, a microfluidics-based solution to enable high-throughput assay development and screening will contribute to improved process efficiency and enhanced productivity across drug discovery and development pipelines. 

is Director of Applications and Technology Assessment at Gyros AB (Sweden).

is Senior Application Scientist Global Collaborations and Support, Gyros AB (Sweden).

Articles

Efficiency is more than just a buzzword in today's pharmaceutical industry; declining productivity and diminishing returns on investment have made it an over-arching mindset that is critical to corporate survival.