Validated assay for Mab concentration

Stricter regulatory requirements have created the need for more rapid and precise validated assays for QC in pharma environments. Biacore describes how this might be done

Manufacturing regulations require that the concentration of proteins, - for example, those produced by recombinant or hybridoma technology - is monitored throughout the drug development and production process, preferably using validated assays. The use of such assays helps to ensure reliable and reproducible results and, therefore, the safety and efficacy of pharmaceutical products. Traditionally, ELISAs (enzyme-linked immunosorbent assays) have been used for protein concentration analysis, but stricter regulatory requirements have created the need for more rapid and precise validated assays for quality control (QC) in pharmaceutical environments.

The development of a validated assay satisfying regulatory GxP requirements may involve the optimisation of individual assay components and the identification of factors that could lead to errors in results. Accuracy, precision, selectivity, sensitivity, reproducibility and stability must all be demonstrated to be of sufficiently high quality before the assay can be considered suitable for its intended use. The validation process can be time-consuming, however, and there is a need for more efficient assay development.

Here, Biacore describes the process leading to the rapid establishment of a validated assay for the determination of monoclonal antibody (Mab) concentration. This work can be regarded as a model system for the development of other similar concentration assays.

Micromet, a biotechnology company based in Munich, Germany, has developed novel antibody-derived therapeutics for the treatment of human disease. One drug candidate, MT201, is designed for the treatment of prostate cancer. MT201 is a fully human antibody targeted at EpCAM (human epithelial cellular adhesion molecule), present on the surface of many tumour cells.¹ Micromet's researchers were able to establish, within only three months, a rapid, validated assay to measure MT201 concentration in harvest samples, process controls and final product using Biacore C, a surface plasmon resonance (SPR) -based instrument, designed for rapid concentration analysis of protein-based therapeutics in drug development, process control applications and manufacturing QC. The instrument and its control software have been developed to comply with current GxP requirements and 21 CFR Part 11.

While there are no definitive guidelines available on the validation of ligand binding data, documents published by the US FDA and ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) provide generally accepted guidelines for binding assay design.^2-4 The assay described here was developed according to these guidelines.

principle of SPR

MT201 concentration determination relies on detecting binding between MT201 and protein A, a molecule with high affinity for IgG, immobilised on the surface of a sensor chip (Sensor Chip CM5, Biacore, Sweden). When a solution containing MT201 is passed over the sensor surface, MT201 binds to protein A, causing a change in analyte concentration close to the surface and generating an increase in the SPR signal (see box on page 47). This signal, which is expressed in Resonance Units (RU), is displayed graphically as a sensorgram and can be related to the concentration of MT201 in the solution.

Parameters such as degree of protein A immobilisation, surface regeneration and sensor chip storage conditions were investigated to determine their effects on MT201 binding activity and to optimise conditions for the final assay design. It was necessary to establish, for example, that the amount of protein A immobilised on the sensor surface was consistent across batches of protein A.

To test this, 16 different lots of protein A at a concentration of 10µg/ml were injected over the sensor surface at a flow rate of 5µl/minute for seven minutes. Immobilisation levels reflected in terms of signals between 1500 and 2500 RU were recorded (mean: 1970 ± 137 RU), demonstrating that there were no significant differences in protein A immobilisation levels when using different batches under the same immobilisation conditions.

A range of MT201 concentrations was then passed over the sensor surface to measure binding levels. Optimal binding of MT201 was achieved using protein A immobilised as described above. No additional MT201 binding occurring when protein A immobilisation levels were increased beyond this level (Figure 1).

A sensor surface can be restored for further analytical cycles using a regeneration buffer. More than 20 buffers were screened in various combinations to select the chemical regime which was least aggressive, but which still provided acceptable surface regeneration (Fig. 2). The chosen buffer was 0.5 M NaSCN containing 10 mM NaOH.

storage variations

It is possible to store pre-immobilised protein A-bound sensor chips but it was necessary to establish whether, and to what extent, storage under different conditions could affect activity. To test this, the researchers stored Sensor Chip CM5 with bound protein A in air or in HBS-EP buffer containing sodium azide (Biacore, Uppsala, Sweden) for periods of up to two months. Surfaces exposed to air lost up to 50% of their activity after one week and up to 70% of their activity after two weeks. In contrast, chips stored in HBS-EP buffer remained active for at least two months (Fig. 3).

Protein A leakage from the chromatography column during MT201 purification is a significant source of possible error in the measurement of MT201 concentration. Up to 50ng/ml of protein A may leach from the column to contaminate the sample. This leached protein A could bind MT201 in solution and prevent binding to protein A on the sensor surface. To measure the possible effects of free protein A, 0.016-25ng/ml protein A solutions were spiked with 2.5ng/ml MT201 and tested using the Biacore C assay. Signal inhibition was only found at protein A concentrations above 0.5ng/ml after 1:500 column eluate dilution, well above the concentrations expected after elution of antibody from the column.

Other possible sources of error were also investigated. It was found, for example, that bovine serum albumin (BSA) in the sample buffer bound competitively to protein A, greatly affecting concentration measurements at low concentrations of MT201. Researchers also found that surfactant concentrations as low as 0.001% in sample buffer had a significant effect on the signal. At concentrations above 0.1%, however, the influence of surfactant levelled out with negligible additional effects across a range of MT201 concentrations up to 10µg/ml.

Inter-assay variability was quantified using 26 assays performed on different days within a period of seven days using four different sensor chips. Variability was below 10% at the highest concentration of MT201 tested.

Intra-assay variability was also measured using different lots of Sensor Chip CM5 and repeated measurements of three different MT201 concentrations added to a sample containing 0.25% surfactant. Variability was less that 5% in samples spiked with 5 µg/ml MT201; below 10% in samples spiked with 1.25 µg/ml MT201; and below 20% in those spiked with 0.08 µg/ml MT201. Intra-assay variability naturally increased close to the limit of quantification, which was determined to be 80ng/ml.

The activity of freshly diluted samples and samples stored at room temperature for 24 hours were compared to verify that short-term storage had no significant effect. Similar tests were performed using samples stored at 37°C, at -80°C for extended periods, and those subjected to five freeze-thaw cycles. Assay performance was not affected.

As ELISAs have been considered the method of choice for concentration determination, it was important to compare Biacore C assay performance with that of ELISA. In a single direct comparison in which identical samples were measured, Biacore C gave both lower standard deviations and lower variances in data than the ELISA (Figure 4). The assay was also faster than the single end point ELISA format.

The concentration assay developed by Micromet's scientists demonstrates the usefulness of Biacore C for rapid assay development in GxP-regulated environments. While the assay developed by Micromet was designed for MT201, Biacore C may be used for the development of other concentration assays in a variety of therapeutic and downstream research areas.