Table 3.

Optimized formulations and predicted responses, DoE1

Factor						Predicted response
Desirability	Disaccharide concentration (%, w/v)	Disaccharide type	Mannitol (%, w/v)	Buffer	pH	Z-averaged (nm)a	Stressed Z-averaged (nm)b	GLA (μg/mL)c	ΔpHd	ΔpH stresse	Cake qualityf
0.68	6.8%	Trehalose	0%	Phosphate	7.5	117	124	44	0	0	1
0.63	10%	Trehalose	0%	Tris	7.5	120	124	47	−0.2	0	1
0.57	8.4%	Trehalose	1%	Phosphate	7.5	122	131	43	0.1	0	1
0.48	8.2%	Trehalose	1%	Tris	7.5	124	132	40	−0.2	0	1
0.66	7.5%	Sucrose	1%	Phosphate	8	117	119	38	0	0	1
0.66	5.7%	Sucrose	0%	Phosphate	7.5	111	128	42	0	−0.1	1
0.54	10.0%	Sucrose	0%	Tris	7.5	112	127	33	−0.1	0	1
0.16	9.9%	Sucrose	1%	Tris	7.5	115	121	29	−0.2	0.1	2

Notes:

^aPredicted Z-average diameter measured after lyophilization and reconstitution.

^bPredicted Z-average diameter measured after heat stress for 30 days with storage at 50°C.

^cPredicted GLA concentration after heat stress for 30 days with storage at 50°C.

^dPredicted change in pH after lyophilization and reconstitution.

^ePredicted change in pH after heat stress for 30 days with storage at 50°C.

^fPredicted cake quality after heat stress for 30 days with storage at 50°C. All lyophilized cakes from DoE were considered acceptable after lyophilization and were designated 1 – no change or 2 – change based on visual observation after heat stress.

Abbreviations: GLA, glucopyranosyl lipid adjuvant; DoE, design of experiments.