Table 1.
Excipient Content in Formulations Prepared for Liquid Stability Screening.
Fig 1.
Stability of Subunit Vaccine in Liquid Formulations at 4°C (left) and 37°C (right).
Formulations were prepared with concentrated bulk H1N1 subunit vaccine (H1N1 California/07/2009 X179A). In addition to the stabilizers shown in the figure, all formulations contain 45 μg HA/mL, 20 mM phosphate buffer, 0.09% polysorbate 80 and 14 mM NaCl (F8 was in PBS which contained 140 mM NaCl). The formulations were stored at 4°C (A, left) and 37°C (B, right), and their HA titers were measured by SRID. Formulations were tested for up to 7 days. For screening, one dilution for each formulation was tested by SRID in triplicate. The data were presented as percentage recovery relative to the mean HA titer after formulation preparation (time 0) ± standard deviation of each triplicate. Abbreviations: SRID, single radial immunodiffusion; HA, hemagglutinin; PBS, phosphate buffer saline.
Table 2.
Composition of Formulations Used in Screening for Freeze-dried H1N1 Subunit Vaccine.
Fig 2.
Stability of Freeze-dried Influenza Vaccine Formulations (Round 1) at 37°C (A) and 45°C (B).
Formulations were prepared with concentrated bulk H1N1 subunit vaccine (H1N1 California/07/2009 X179A). In addition to the stabilizers shown in the figure, all formulations contain 45 μg HA/mL, 20 mM phosphate buffer, 0.09% polysorbate 80 and 14 mM NaCl. The formulations were freeze-dried as described in Materials and Methods and stored at 37°C (A, left) and 45°C (B, right). The HA titers were measured by SRID up to three months. For screening, one dilution for each formulation was tested by SRID in triplicate. The data were presented as percentage recovery relative to the mean HA titer after formulation preparation but before freeze drying (Pre-lyo) ± standard deviation of each triplicate. Abbreviations: SRID, single radial immunodiffusion; HA, hemagglutinin.
Fig 3.
Stability of Freeze-dried influenza vaccine Formulations (Round 2) at 37°C (A) and 48°C (B).
Formulations were prepared with concentrated bulk H1N1 subunit vaccine (H1N1 California/07/2009 X179A). In addition to the stabilizers shown in the figure, all formulations contain 45 μg HA/mL, 20 mM phosphate buffer, 0.09% polysorbate 80 and 14 mM NaCl. The formulations were freeze-dried as described in Materials and Methods and stored at 37°C (A, left) and 48°C (B, right). The HA titers were measured by SRID up to two months. One dilution for each formulation was tested by SRID in triplicate. The data were presented as percentage recovery relative to the mean HA titer after formulation preparation and before freeze-drying (Pre-Lyo) ± standard deviation of each triplicate. Abbreviations: SRID, single radial immunodiffusion; HA, hemagglutinin.
Fig 4.
Moisture Contents of all Formulations in Rounds 1 and 2.
Water in freeze-dried formulations was extracted with anhydrous methanol and then determined by Karl Fischer coulometry. The percentage of water content in the dried powder (w/w) was calculated by comparing the amount of extracted water to the weight of the lyophilized vaccine formulation. Three vials were measured for each formulation. The data were represented as the mean of the three samples ± standard deviation.
Table 3.
Composition of Selected Freeze-dried Lead Formulations for Long-term Stability Study.
Fig 5.
Long-term Stability of Freeze-dried Lead Formulations of H1N1 Subunit Vaccine Stored at 4°C, 25°C, and 37°C.
Two freeze-dried lead formulations were prepared with concentrated bulk H1N1 subunit vaccine (H1N1 California/07/2009 X179A) in phosphate buffer and additional excipients listed in Table 3. The liquid formulation obtained from dilution of bulk vaccine with PBS was used as the control. The stability at 4°C (A), 25°C (B), and 37°C (C) was monitored up to 40 months. For each formulation, three vials were reconstituted and combined. Two dilutions were prepared within the linear range of the standard curve and tested by SRID in triplicate. The data represented the mean HA titer ± standard deviation of six measurements.
Fig 6.
Residual Moisture Content of Freeze-dried Lead Formulations of H1N1 Subunit Vaccine Stored at 4°C, 25°C, and 37°C.
The moisture content in stability samples of two freeze-dried lead formulations, stored at 4°C, 25°C, and 37°C, was monitored for three years by using Karl Fischer coulometry. The percentage of water content in the dried powder (w/w) was calculated by comparing the amount of extracted water to the weight of the lyophilized vaccine formulation. Three vials were measured for each formulation at each time point. The data was represented as the mean of the three samples ± standard deviation. Due to the limited number of sample vials, only two vials at week 104 and one vial at week 156 were tested. For the sample stored at 4°C, only one vial was tested at week 12.
Fig 7.
HAI Titers in Mice Immunized with H1N1 (A/Cal/07/2009) Influenza Subunit Vaccine.
Each study group consisted of 10 mice. Animals were immunized intramuscularly with 100 μL of either reconstituted freeze-dried H1N1 subunit vaccine or liquid vaccine diluted to 0.1 μg/mL (upper panel: A) and 1.0 μg/mL (lower panel: B) and blood samples were collected prior to vaccination (weeks 0 and 4) and 4 weeks after the final vaccination (week 8). HAI tiers in serum samples against influenza A/Cal/07/2009 were measured in duplicate, at week 0 (left, pre-immunization), week 4 (middle) and week 8 (right), and were expressed as the reciprocal of the highest dilution demonstrating HA inhibition. Data of individual mice are shown. Geometric mean titer of each group is shown by a horizontal bar. HAI titers induced by immunization at week 4 and week 8 were significantly different from HAI titers at baseline (week 0), (p≤0.002 for groups immunized with 0.01 μg and p<0.05 for groups immunized with 0.1 μg.
Fig 8.
Overlay of X-ray Powder Diffractograms of Freeze-dried Powder.
X-ray powder diffraction (XRPD) patterns of freeze-dried powders of three batches of Lead 1 and Lead 1 controls with sucrose only and glycine only were determined with Rigaku SmartLab system at Triclinic Labs (Lafayette, IN) under low-humidity conditions, as described in Materials and Methods.
Table 4.
The Ratio of Crystalline to Noncrystalline Structure Based on X-ray Diffraction.