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The development of biotechnology-derived pharmaceuticals is a stepwise process involving an evaluation of both animal and human efficacy and safety information. Before a biotechnological or biological product is studied in humans, a precise and comprehensive characterization of the product should be conducted and described. Performing proper preclinical safety evaluation can reduce the time and cost for approval of new biopharmaceuticals.

The assessment of the biological activity that defines the specific ability or capacity of a product to achieve a specific biological effect is essential. Comparisons of target sequence homology between species can be an appropriate starting point when determining species relevancy and in-vitro assays can be used to predict specific aspects of in-vivo activity and may be designed to assist in the selection of appropriate animal species.

A relevant species is one in which the product is pharmacologically active, the receptor or an epitope (in the case of monoclonal antibodies) is present or expressed and tissue cross-reactivity profiles are similar to humans.

For animal model selection, biotherapeutics product binding information can be very helpful. Surface Plasmon Resonance (SPR) is a powerful technique to measure biomolecular interactions and can be used to qualitatively/quantitatively demonstrate cross-species comparisons regarding binding affinities and/or kinetics.

Methods

Our laboratory utilises the BiacoreTM T200 system (GE Healthcare), using SPR technology, to perform biomolecular interaction analysis. The binding protein (proprietary) was immobilised onto the Biacore Sensor Chip surface and considered as the ligand. The binding partners, considered as analytes, were flowed over the immobilised ligand and the real time binding response was assessed. A reference surface and a zero concentration were included for double reference subtraction.

The analysis was performed at 25°C in HBS-EP+ buffer at a flow rate of 30 µL/min and analyte concentrations of 1.25, 2.5, 5, 10, 20 (in duplicate) and 40 nM were considered. For all the injected analyte concentrations, a constant association period of 300 seconds and a dissociation period of 2100 seconds were applied followed by a regeneration of the surface with a solution of glycine.

Results

In the example described here (Figure 1), we have assessed the interaction of a human binding protein with its known human binding partner and homologues from different species: monkey, rabbit, mouse, rat and dog.



Similar association and binding responses can be observed for human, monkey and rabbit binding partners, with a slightly smaller response for rat, however no binding is observed with dog. This can be due to an inactive product (e.g. misfolded proteins, masked binding site by the purification tag) or where the binding protein didn’t interact per se with the considered partner.

The binding detected with the rat binding partner is very stable, in a similar way to human where only little dissociation is observed. On the other hand, the binding response of rabbit presents a slightly faster dissociation in comparison to that of human or rat binding interaction.

The kinetics data, obtained from a minimum of triplicate analysis, are summarised in an On-Off Rate Map (Figure 2) and are presented in Table 1.

graph 

kinetic data
The binding kinetics and affinity of the monkey and human binding partners are very similar, whereas the binding of rabbit is slightly different with a slower association and dissociation rate resulting in a lower affinity.

The binding kinetics for the mouse and rat binding partners cannot be defined with this experiment configuration, as the interaction is very stable, reaching the limits of detection of the equipment. However, from the data collected it can be predicted that the affinity for the binding to the human binding protein is in the picomolar range.

Conclusion

The BiacoreTM T200 can provide reliable information on product binding properties and can help highlight slight difference in the kinetics and/or affinity with the binding partner from different species, as shown in the selected example.

Preclinical results, as demonstrated in this document, can help product manufacturer to justify the selection of a relevant animal model species by providing important information on model similarity.

For further information, please contact:

Melanie Verneret
Molecular Bioanalytical Scientist
SGS Life Science Services
5 South Avenue
Clydebank Business Park
Glasgow, United Kingdom
t: +44 (0)141 952 0022

References:

  1. FDA Guidance for Industry S6 Preclinical Safety Evaluation of Biotechnology-derived pharmaceuticals
  2. FDA Guidance for Industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals
  3. FDA Point to consider in the manufacturing and testing of monoclonal antibody products for human use
  4. Guideline on development, production, characterization and specifications for monoclonal antibodies and related products EMEA_CHMP_BWP_157653_2007