Effect of formulation on the stability and aerosol performance of a nebulized antibody

Figures & data

Table 1. Characteristics of a 10 mg/ml solution of antibody in PBS pH 7.2 after nebulization with three nebulizers. Each result is the mean (± SD) of three nebulizations

Nebulizer	Hydrodynamic diameter (nm ± SD)	Percentage monomers (% mass)	Size exclusion chromatography (% monomers/% other aggregates)	VMD (μm) (mean ± SD)	Flow rate (ml/min ± SD)
Omron	NA	NA	91.1 ± 0.6 / 8.9 ± 0.5	5.6 ± 0.1	0.23 ± 0.04
Aerogen Solo	NA	NA	91.3 ± 2.5 / 8.7 ± 1.9	4.1 ± 0.1	0.50 ± 0.01
PARI eFlow	NA	NA	90.6 ± 0.5 / 9.5 ± 0.6	3.8 ± 0.3	0.45 ± 0.05

Figure 1. Large antibody aggregates were analyzed by fluorescence microscopy before and after nebulization with the three nebulizers (Omron, PARI eFlow, Aerogen Solo), for a 10 mg/ml antibody solution.

Figure 2. A and 2B. Size exclusion chromatography of a 10 mg/ml antibody solution in PBS before (solid line) and after (dotted line) nebulization with the Aerogen Solo. (A) UV signal; (B) MALLS signal. The left is Molecular Weight determined by MALLS and the right axis is light scatter in arbitrary units.

Figure 3. The percentage increase in the number of large antibody aggregates was analyzed by fluorescence microscopy following nebulization, for the three nebulizers (Omron, PARI eFlow, Aerogen Solo).

Table 2. Minimum concentration of PS-20 required to prevent the production of medium-sized aggregates during the nebulization of an antibody solution (10 mg/ml in PBS pH 7.2) with three nebulizers

Nebulizer	Minimum PS-20 concentration (% w/v)
Omron	10^−5
Aerogen Solo	10^−4
PARI eFlow	10^−3

Table 3. Characteristics of a 10 mg/ml antibody solution in PBS pH 7.2 supplemented with various concentrations of PS-20, after nebulization with the Aerogen Solo nebulizer. Each result is the mean (± SD) of three nebulizations

PS-20 concentration% (w/v)	Hydrodynamic diameter (nm ± SD)	Percentage monomers (% mass ± SD)	VMD (μm) (mean ± SD)	Flow rate (ml/min ± SD)	Viscosity (mm^2/s ± SD)
0%	NA	NA	4.1 ± 0.1	0.50 ± 0.01	1.07 ± 1.81E-02
0.00001%	NA	NA	3.8 ± 0.5	0.54 ± 0.04	1.05 ± 7.34E-03
0.0001%	NA	NA	3.8 ± 0.2	0.49 ± 0.01	1.03 ± 5.23E-03
0.001%	11.4 ± 0.5	99.9 ± 0.2	3.6 ± 0.6	0.49 ± 0.01	1.03 ± 2.00E-03
0.01%	11.2 ± 0.3	99.9 ± 0.2	3.8 ± 0.4	0.48 ± 0.01	1.02 ± 5.46E-03

Table 4. Characteristics of solutions of two monoclonal antibodies (IgG1 and IgG4) in different buffers with various concentrations of polysorbate, before and after nebulization. Each result is the mean (± SD) of three nebulizations

				DLS (% of monomer ± SD)		Large antibody aggregates (10^3/ml ± SD)
	Nebulizer	Type of surfactant	Surfactant concentration (% w/v)	Before nebulization	After nebulization	Before nebulization	After nebulization
Mab 1 – Buffer 1*	Aerogen solo	PS-20	0	99.6	99.1	85.6	128.8
			0.01	99.2	98.6	27.9	45.9
			0.1	99.9	100	33.3	29.7
Mab 2 – Buffer 2**	PARI eFlow	None	0	100	NA	918.0	NA
		PS-20	0.01	100	97.3 ± 3.8	607.5	1024.7 ± 356.4
			0.1	100	99.6 ± 0.3	783.0	625.5 ± 232.4
		PS-80	0.01	100	99.4 ± 0.4	388.8	954.0 ± 623.5
			0.1	100	99.9 ± 0.0	702.0	688.5 ± 269.0

* n = 1 nebulization; **n = 3 nebulizations; NA, not analyzable.

Table 5. Characteristics of solutions of various concentrations of antibody in PBS pH 7.2, after nebulization with the Aerogen Solo nebulizer. Each result is the mean (± SD) of three nebulizations

Antibody concentration (mg/ml)	Hydrodynamic diameter (nm ± SD)	Percentage monomers (mean% mass)	VMD (μm) (mean ± SD)	Flow rate (ml/min ± SD)	Viscosity (mm^2/s ± SD)
2	NA	NA	4.4 ± 0.3	0.55 ± 0.01	0.97 ± 1.02E-03
5	NA	NA	3.6 ± 0.2	0.48 ± 0.01	1.02 ± 4.40E-03
10	NA	NA	4.1 ± 0.1	0.50 ± 0.01	1.07 ± 1.81E-02
15	12.6 ± 1.42	94.4 ± 9.0	4.0 ± 0.1	0.47 ± 0.01	1.09 ± 2.00E-02
30	11.9 ± 0.06	99.6 ± 0.2	4.1 ± 0.1	0.42 ± 0.01	1.17 ± 7.38E-03
40/45	12.6 ± 0.35	99.6 ± 0.5	4.0 ± 0.2	0.40 ± 0.01	1.28 ± 9.97E-03

Figure 4. Large antibody aggregates were analyzed by fluorescence microscopy before and after nebulization with the Aerogen Solo nebulizer, for various antibody concentrations.

Figure 5. Model of the spatial distribution of antibodies in aerosol droplets as a function of antibody concentration.

Table 6. Minimum concentration of PS-20 required to limit the formation of medium-sized and large aggregates

	Antibody concentration (mg/ml)
	2	5	10	15	30	40
Minimum PS-20 concentration (% w/v)	0.001	0.0001	0.0001	0.0001	0	0