(M1330-11-70) A Novel Approach to Formulation Screening of High Concentration Recombinant Human Heparan-N-Sulfatase (rhHNS) Using High-Throughput Devices

Purpose: Mucopolysaccharidosis type IIIA (MPSIIIA) is a pediatric metabolic neuropathology caused by the inherited deficiency of the lysosomal enzyme sulfamidase. MPSIIIA represents one of the most common forms of lysosomal storage disorders and there is no known cure. To find a cure to MPSIIIA, we plan to break down GAGs accumulated in the brain by administering the drug directly into the cerebral ventricle, instead of crossing the blood-brain barrier. For a study of this nature, neurotoxicity is commonly observed as the main side effect of cerebral ventricle drug delivery, arising from a variety of factors, such as unsuitable excipients and even the active drug itself. For this reason, cerebral ventricle formulations should ideally contain as few excipients as possible and the active drug must be screened for its propensity to cause neurotoxicity. In this study, we screened formulation for suitable excipients in order to maintain safety and stability for the brain. The formulation study was carried out adhering to these considerations.

Methods: 1) The formulation screening focused on investigating particle interaction parameters, B₂₂, as predictors of protein aggregation kinetics by using an Uncle high-throughput device. In this study, the buffer agents for the histidine, glutamate and arginine were fixed at a concentration of 20 mM in a 200 mM NaCl solution. The samples was prepared at concentrations of 2.52, 5.04, 7.56, 10.08, and 12.6 mg/ml, and the 8.8 uL per sample was loaded to the Uni consisting of 16x9 uL micro cuvettes. 2) Protein concentration and turbidity were detected by a Lunatic high-throughput device. For this analysis, the samples were prepared at concentrations of 15 mg/ml, and the 2 uL samples were loaded into the high lunatic chip. All of samples were detected at 280, 330 and 350 nm wavelength. 3) Sub-visible and visible particles were measured by an AURA high-throughput device. For this analysis, the samples were prepared at concentrations of 15 mg/ml and the 50 uL samples were loaded on the 96 plate well.

Results: At the beginning of this experiment, 20 mM solutions of histidine, glutamate, and arginine were screened to maintain the buffering capacity and stability of their HNS protein. A histidine sample containing 12.6 mg/ml HNS obtained a positive B₂₂ value(0.0000591 molᆞmL/g²) and the other samples obtained negative B₂₂ values. A positive B₂₂ value indicates that solutes prefer association with the solvent over self-association due to strong electrostatic repulsion between solute molecules. Furthermore, The Uncle P.D.I(Poly Disperse Index) result of a histidine sample was 0.0063. This shows that the histidine sample was monodispersed, despite containing a 12.6 mg/ml concentration of HNS. However, the P.D.I results of the other samples obtained a value greater than 0.3 which indicates that those samples were polydispersed. The pH screening was performed for all samples between a pH of 7.5 and 9.0, with consideration to the HNS P.I. values. Our data indicated that pH value is the main factor contributing to HNS protein solubility. The pH value of the formulation buffer affects both appearance and turbidity. The sample showed a lower turbidity when the pH value is over 8.00. The turbidity results were 1.654, 0.947, 0.143, 0.057, 0.045 and 0.067 when the pH values were 7.53, 7.70, 8.00, 8.17, 8.27 and 8.45. These results indicate that it could be possible to condense a higher concentration of the HNS protein when the pH of the buffer is over 8.00. Additionally, dependent histidine treatment at a pH of 8.00 could enhance the solubility of the HNS protein whilst remaining brain safe. Lastly, the MPSIIIA drug product is a lyophilized product, and is used after undergoing a reconstitution process in which a PS20 solution is added for the purpose of enhancing stability and reducing visible and sub-visible particles of the drug product. For this study, sub-visible and visible particles were measured by an AURA high-throughput device. Through the use of this device, we were able to predict the number of particles in the samples and also determine the count of self-aggregated HNS proteins in different formulation buffers.

Conclusion: Finally, we investigated the MPSIIIA formulation screening study and were able to select the excipients which provided the most improvements in protein stability and brain safety via the use of high-throughput devices. This research could contribute to the development of high concentration HNS drug products. For pediatric patients who need to receive their drug regularly, these drug products allows for a less frequent dosing and reduces a risk by injecting a small volume to the brain.

References: 1) Brain Targeting in MPS-IIIA., Fraldi A
2) Intrathecal route of drug delivery can save lives or improve quality of life., Kalliopi Dodou