(M1530-03-13) ALD Nano Coating on Lyophilized and Spray Dried Biologics: Feasibility and Stability Improvement

Purpose: The therapeutic protein can undergo various chemical and physical instabilities in aqueous solutions during storage, which need to be minimized. An approach to enhance protein stability is the removal of water^1,2,3 to achieve longer and commercially viable shelf lives. Atomic layer deposition (ALD) has demonstrated multiple benefits for small molecules including processability, stability, shelf life, release control⁴, etc. ALD is a solvent-free, ambient temperature, layer by layer coating technique, building a nanoscale film ( < 10-100 nm) on the surface of particles. The surface engineering with ALD on amorphous solid dispersion successfully inhibits the crystallization of API over the period of 2 years under acceleration conditions^5,6. In this study, we studied the impact of the ALD process on stability regarding proteins’, antibodies’ structure and functionality in solid form, a potential approach to eliminate cold chain requirement for biologics.

Methods: Lyophilized Avastin (Bevacizumab) and trypsin were selected to investigate the ALD impact on structure and functionality. Spray-dried and lyophilized myoglobin with mannitol (1:1 wt% ratio) mixture was selected for the stability study, the reason for selecting mannitol over trehalose as the excipient is to aim a quick degradation for comparison of ALD coated and uncoated powders. Fourier-transform infrared spectroscopy (FTIR), size-exclusion chromatography (SEC), intact mass analysis, peptide mapping, and fluorescence were conducted on the powders before and after ALD process. Enzymatic assay of trypsin was done with N-benzoyl-L-arginine ethyl ester (BAEE) as substrate as per USP (United States Pharmacopoeia) monogram.⁷

Results: Avastin (bevacizumab) was coated with Titanium oxide through the ALD process. No structural changes (primary, secondary, tertiary) were observed after the ALD process (see Figure 1). This demonstrates the ALD surface nano engineer process is a feasible process on protein and antibody, without impacting protein structure. Trypsin molecular weight is ~150 K Daltons. Aluminum oxide and zinc oxide material are coated on trypsin with two different thicknesses. Based on the enzymatic assay test, trypsin activity was found to meet USP specification, indicating that ALD coating of aluminum oxide and zinc oxide didn’t show any impact on the functionality of trypsin (Figure 1). SEM images of aluminum oxide ALD coated myoglobin/mannitol particles from spray drying and lyophilization processes showed no morphology change after ALD process (Figure 2A, 2B). ALD coating thickness is 8.3 nm from TEM cross section image (Figure 2C). The ALD-coated myoglobin/mannitol particles transform the fluffy, loose lyophilization powder into dense and free-flowing powders (Figure 2D, 2E). Summarized myoglobin/mannitol powder’s performance before and after the ALD process listed in Figure 3. Aggregation and reconstitution time were within the approved limit, moisture content further reduced to < 1%. Aggregation and monomeric loss before and after 3 months under accelerated testing conditions of 40^°0C, 53% RH were depicted in Figure 3. ALD coating showed < 5% aggregation, around 2% monomeric loss for spray-dried myoglobin powders and 6-8% for lyophilized powder, uncoated myoglobin shows 20-50% aggregation and monomeric loss⁸.

Conclusion: The present study shows spray drying is the preferred technique to convert liquid biologics into solids due to well-controlled particle size distribution and better morphology than that of lyophilized powder. ALD process is compatible with biologics. The coating process doesn’t damage the physical structure, functionality of proteins, or antibodies. Initial POC studies showed that the coating improves the stability of biologics with maintain less than 5% aggregation under acceleration conditions, indicating a potential approach to eliminate cold chain storage and shipment requirements, the huge problem for the current biotherapeutic industry. Like our demonstration on small molecules, the ALD-coated biologic powders show good flowability and density ⁴, which is beneficial to the post-manufacture process like vial filling.

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Acknowledgements: The authors are thankful to Prof Anurag S. Rathore, IIT Delhi for the collaborative work on Avastin and Prof Elizabeth Topp, National Institute for Bioprocessing Research and Training (NIBRT), Dublin, Ireland for the collaborative work on myoglobin, and BioGenomics Limited, India for collaborative trypsin work.


Figure 1: Structure characterization of ALD coated Bevacizumab: intact-MS analysis, Peptide mapping, FTIR and fluorescence; trypsin enzymatic assay test results for ALD coated and uncoated trypsin.


Figure 2: SEM of ALD coated myoglobin/mannitol powder (A, B) and TEM cross section of aluminum (8 nm) coated powder (C), Morphology of uncoated (D) and coated (E) of lyophilized myoglobin/mannitol powders.


Figure 3: TOP: Myoglobin properties comparison before and after ALD process at t=0. (SD: spray dried powders; Lyo: lyophilized powders) Bottom: Aggregation % of Myoglobin (Left); loss of monomeric peak area % of myoglobin (right) at t=0 and 3 month acceleration test under 40C, 53% RH.