. 2012 May 14;287(30):25266–25279. doi: 10.1074/jbc.M111.330902

TABLE 2.

Particle characteristics (2–10 μm size; partially folded conformation) correlate with the induction of the innate immune response

Molecule	Stress treatment	Response of PBMC^a	Particle concentration^b			Folded secondary structure^c	p value^d
Molecule	Stress treatment	Response of PBMC^a	<2 μm	2–10 μm	>10 μm	Folded secondary structure^c	p value^d
						%
mAb1	microspheres	+++++^e	–	+++++	ND^f	70^g	***
	stir-20h	++++	++++	++++	++	42	***
	stir-3d	+++	+	+++	+++	20	*
	65C/pH 8.5	+++	++	++++	+++	26	***
	syringe-so⁺	+++	++++	+++	++	32	*
	monomer	++	+	+	ND	100	**
mAb2	stir-20h	+++++	–	++++	++	46	***
	stir-3d	++++	–	++++	+++	33	***
	65C/pH 8.5	+++	–	++++	++++	12	***
	syringe-so⁺	+	–	+++	++	49	/
	monomer	+	–	++	+	100	/
mAb3	stir-20h	++++	++++	++++	++	–	***
	stir-3d	+++	++	++++	++	–	**
	65C/pH 8.5	+++	+	+++	++++	–	/
	syringe-so⁺	+++	++++	+++	+++	–	/
	monomer	ND	+	+	+	100	/

^a The relative response of PBMC from 22 donors at the innate phase to mAb1, mAb2, and mAb3 monomer or aggregated samples is shown. The + symbol indicates the relative strength of the aggregate-induced response in PBMC as measured by the magnitude of the response and the number of donors that responded (Fig. 2).

^b Particle concentration in discrete size ranges was determined by nanoparticle tracking analysis (<2 μm) or HIAC (2–10 μm; >10 μm). The + symbol indicates the relative fold increase (26).

^c Correlation coefficient (%) between the spectra of the monomeric mAb and pellet fraction of the aggregated mAb is shown (26).

^d p values generated by a mixed effect statistical model were used to indicate statistically significant differences between 22 donors treated with aggregated mAbs as compared with the background response. MCP-1 was used as a representative cytokine. Eight donors were used for the microsphere calculation. Statistically significant increases are denoted with one asterisk (p value < 0.048), two asterisks (p value < 0.010), or three asterisks (p value < 0.001). / indicates no significant differences (p value >0.048).

^e + indicates the relative fold increase or extent of change, and – means not tested.

^f ND means not detected.

^g Average of several analyses is shown. Data showed variability due to low concentration of protein on the mAb1-microspheres and because the microspheres themselves have a spectra. All of the mAb1 aggregates, but not the mAb1-microspheres, displayed a shift in the minima compared with the mAb1 monomer spectra.