(M1330-03-15) Effect of Atomic Layer Coatings on the Stability of Solid Myoglobin Formulations

Purpose: Therapeutic proteins may suffer from instabilities such as denaturation or aggregation due to thermal, mechanical, or chemical stress. Proteins are often less susceptible to degradation in the solid state, although solid-state stability can be affected by the formulation, drying method, and storage conditions. Here, the stability of myoglobin in the solid-state was assessed following Atomic Layer Coating (ALC), a promising process long employed in semiconductor manufacturing but seldom studied in biopharmaceuticals. ALC produces a thin film of organic or inorganic material on a solid surface forming a uniform coating. This study tested the hypothesis that aluminum oxide and/or silicone oxide ALC coating reduces myoglobin aggregation during storage in lyophilized or spray dried powders relative to uncoated controls while providing acceptable reconstitution time.

Methods: Powders containing myoglobin and mannitol (1:1 w/w) were generated from a solution containing myoglobin (10 mg/mL) and mannitol (10 mg/mL) in potassium phosphate buffer (2.5 mM, pH 7.0) by either lyophilization or spray-drying, and then subjected to ALC with aluminum oxide or silicon oxide coating. After coating, the formulations were stored for 3 months under controlled conditions (53% RH, 40 ^oC) in open or closed vials. After the storage period, reconstitution time was measured, and moisture content was determined by coulometric Karl Fisher titration. Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) was performed to assess protein recovery, percentage of soluble aggregates, and total monomer peak loss.

Results: In all cases, ALC resulted in lower moisture content (for example, lyophilized and spray-dried coated samples presented ~1% of water content in comparison with ~5% in uncoated sample controls), and after storage, coated open vial samples showed considerably higher moisture content (from 3 to 5%) than the corresponding samples incubated in closed vials (from 0 to 2%). In most cases, the addition of coating was associated with an increase in the reconstitution time, which was greater after three months of storage (t₃), however, all closed vials reconstituted in less than 6 minutes. Fractional protein recovery was defined relative to an uncoated control formulation of the same type, and values greater than one indicate better protein recovery. Most lyophilized and spray dried coated samples showed fractional recovery values greater than 1.0, and values of 2.0 or greater were achieved in some cases, indicating that fractional recovery was better for coated samples than for controls. The percentage soluble aggregates assigned to high molecular weight species (%HMWS) was determined in selected samples with fractional protein recovery not significantly different from 1.0 at t₀ and > 1.5 at t₃ in either open vials or closed vials. After three months of storage lyophilized closed samples with 10, 20, and 40 cycles of aluminum oxide coating presented 9.16, 6.77, and 7.40% of soluble aggregates respectively in comparison with 14.6% in controls. Spray-dried closed samples with 20 cycles of aluminum oxide coating or silicon coating presented 4.82% and 17.15% of soluble aggregates respectively in comparison with 27.5% in controls. Conversely, samples stored in open vials showed values similar to or worse than the controls (Fig. 1). Total monomer loss after storage is the sum of losses due to incomplete recovery and losses due to the formation of soluble aggregates. These losses were least for spray-dried samples with 20 cycles of aluminum oxide coating stored in closed vials (~3%), a marked improvement in the ~50% monomer loss observed for uncoated controls. Lyophilized samples coated with aluminum oxide ALC also showed low monomer loss (~5%), an improvement over the ~25% monomer loss in the uncoated controls (Fig 2). The results showed correlation between monomer loss and moisture content (r²:0.56). Mass spectrometric analysis of coated samples showed no detectable myoglobin chemical degradation and no detectable protein-coating adduct formation resulting from the coating process.

Conclusion: Overall, the results demonstrated that the recovery of soluble, monomeric myoglobin could be improved by ALC coating, and depended on coating type, drying method, and storage conditions. Powders with ALC-coated surfaces had up to 2-fold greater protein recovery than uncoated controls, a result attributed to the effects of ALC coating on powder moisture content and to the inhibition of mannitol crystallization at the particle surface.

Figure 1. Fraction soluble aggregates (%HMWS) for lyophilized (A) and spray dried (B) samples after 10 (Al10), 20 (Al20), or 40 (Al40) cycles of aluminum coating, and silicon coating (Si) at initial time (t0), after 3 months storage in closed vials (t3 closed), and after 3 months storage in open vials (t3 opened). Mean percent high molecular weight species (%HMWS) ± standard deviation, n = 3. * p < 0.05 in one-way ANOVA with Dunnett’s Multiple comparison test indicates significant difference from the Control at the equivalent time and storage conditions (t0, t3 closed, or t3 opened).

Figure 2. Total monomer peak loss detected by SEC; lyophilized myoglobin-mannitol formulations (A), and spray dried myoglobin-mannitol formulations (B) after 10 (Al10), 20 (Al20), or 40 (Al40) cycles of aluminum coating, and silicon coating (Si) at initial time (t0), after 3 months storage in closed vials (t3 closed), and after 3 months storage in open vials (t3 opened). Mean loss ± standard deviation, n = 3. * p < 0.05 in one-way ANOVA with Dunnett’s Multiple comparison test indicates significant difference from the Control in the equivalent times t0, t3 closed, or t3 opened.