Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice

Figures & data

Figure 1. Size distribution of unstressed (Unst), freeze-thawed (FT), pH-shifted (pH), heated (Heat), shaken (Shake) and metal-catalyzed oxidized (Metal Ox) IgG formulations: (A) SEC with UV detection at 280 nm; (B) SEC with MALLS detection and the estimated molar mass of each peak; (C) submicron particles (determined by NTA), (D) micron-sized particles (determined by LO).

Table 1. AUC percentages from SEC-UV analysis and particles/ml of NTA and LO measurements of unstressed and stressed IgG formulations

	SEC-UV (%)						NTA	LO
	Fragments		Monomers	Oligomers	Relative recovery	Total aggregation	Aggregates/ml	Aggregates/ml
Unst	0.7	98.0		1.2	100	1	1 × 10^7	3.0 × 10^3
FT	0.5	98.0		1.4	94	7	4 × 10^7	8.9 × 10^4
pH shift	1.7	49.8		48.5	63	67	3 × 10^9	1.7 × 10^5
Heat	1.0	64.2		34.8	94	39	1 × 10^10	1.6 × 10^4
Shake	2.0	96.6		1.4	12	88	7 × 10^7	4.1 × 10^5
Met Ox	35.8	46.6		17.6	N/A	N/A	7 × 10^7	1.3 × 10^4

Abbreviations and notes: Unst, unstressed; FT, freeze-thawed; Met Ox, metal-catalyzed oxidized; N/A, not available (see text). Percentages of fragments, monomers and oligomers refer to the total AUC of each sample. Relative recovery refers to total AUC of each sample compared with total AUC of the unstressed one. Total aggregation refers to the sum of unrecovered protein plus recovered oligomers.

Figure 2. Representative microscopy images (20x amplification) of unstressed (Unst), freeze-thawed (FT), pH-shifted (pH), heated (Heat), shaken (Shake) and metal-catalyzed oxidized (Metal Ox) IgG formulations after filtration through a 0.22 μm filter, followed by staining with Coomassie brilliant blue. *The filtered volume used for the shaken formulation was 10 times lower than that for the other formulations due to filter blockage.

Figure 3. Structural characterization of unstressed (Unst), freeze-thawed (FT), pH-shifted (pH), heated (Heat), shaken (Shake) and metal-catalyzed oxidized (Metal Ox) IgG formulations by (A) far-UV CD, (B) near-UV CD and (C) Bis-ANS fluorescence.

Figure 4. SDS-PAGE gels of unstressed (Unst), freeze-thawed (FT), pH-shifted (pH), heated (Heat), shaken (Shake) and metal-catalyzed oxidized (Metal Ox) IgG formulations stained with Coomassie brilliant blue under (A) non-reducing and (B) reducing conditions. Dot blots of the same formulations detected with (C) monoclonal anti-human κ light chain antibody (D) polyclonal anti-human heavy chain antibody.

Table 2. Summary of physicochemical characteristics of unstressed and stressed IgG formulations

	Frag	Aggregates					Conform. changes		Hydroph	Covalent bonds	Epitope damage
		Olig	Submicron			Micron	Second	Tertiary
Unst	-	-		-	-		-	-	-	-	-
FT	-	-		+	++		+	+	-	-	-
pH shift	-	++		++	+++		+	++	++	-	-
Heat	-	+++		+++	+		+	+	+++	++	-
Shake	-	-		+	+++		+	N/A	+++	++	-
Met Ox	++	+		+	+		+	++	+	-	-

Abbreviations: Unst, unstressed; FT, freeze-thawed; Met Ox, metal-catalyzed oxidized; Frag, fragments; Olig, oligomers; Conform, conformational; Second, secondary; Hydroph, hydrophobicity; N/A, not available (see text). Explanation of used symbols: negligible (-); mild (+); considerable (++); extensive (+++).

Figure 5. ADA titers of the sera of non-transgenic (NTG) and transgenic (TG) mice treated with unstressed (Unst), freeze-thawed (FT), pH-shifted (pH), heated (Heat), shaken (Shake) and metal-catalyzed oxidized (Metal Ox) human IgG formulations. Time points before the first injection (week 0), during the injection period (week 3 and 5) and after the last injection (week 7 to 16) are shown. The titers are from responders only and the error bars represent the standard deviation of the mean values.