Charge variants in IgG1

Figures & data

Figure 1 Chromatographic profiles obtained from a (A) IEC shown in full scale and (B) SEC shown in expanded scale for all charge variant fractions, starting material and buffer blanks. See Table 2 for numerical values obtained from these analyses.

Figure 2 Representative sensorgrams of fitted data from the kinetic analysis of all charge variant fractions (A) acidic, (B) main, (C) basic and (D) starting material to immobilized rat FcRn. Black lines are fitted curves using a bivalent binding model. All sample concentrations (from bottom to top) are 62.5, 125, 250, 500, 1,000, 2,000 and 4,000 nM. Residual plot shows the difference between experimental and fitted data for every point in the sensorgram. Also see Table 4 for clarity.

Figure 3 Linear changes in serum concentrations versus time for all charge variant fractions following single (A) intraveneous or (B) subcutaneous administration of 10 mg/kg in normal rats. The assay minimum quantifiable concentration was 0.4 µg/mL in rat serum. Results are mean ± SD (n = 12 rats/time point).

Table 1 Major chemical degradation pathways which are a common source of charge-related heterogeneity of therapeutic IgG1 mAbs

Major chemical degradation pathways	Effect	Species formed	References
Sialylation	COOH addition	Acidic	18
Deamidation	COOH formation	Acidic	18, 19
C-terminal lysine cleavage	Loss of NH2	Acidic	18, 20, 21
Adduct formation	COOH formation or loss of NH2	Acidic	18, 19, 22, 23
Succinimide formation	Loss of COOH	Basic	16
Methionine, cysteine, lysine, histidine, tryptophan oxidation	Conformational change	Basic	20
Disulfide-mediated	Conformational change	Basic	24, 25
Asialylation (terminal Galactose)	Loss of COOH	Basic	26
C-terminal lysine and glycine amidation	NH2 formation or loss of COOH	Basic	27

Data mainly adapted from references 16, 18–27.

Table 2 Analytical results to assess all mAb charge variant fractions

Test materiala	Isoelectric pH (pl) (n = 4)b	Protein concentration (mg/mL)	osmolality (mOsm/kg)	Endotoxin (EU/mg protein)	Ion Exchange Chromatography (IEC)			Size Exclusion Chromatography (SEC)
					% acidic	% main	% basic	% aggregate	% monomer	% fragment
Acidic Peak	8.7–8.9c	29.8	161	0.09	95.0	5	NDd	0.3	98.9	0.9
Main Peak	9.0c	30.7	162	0.01	4.4	93.5	2.2	0.1	99.9	NDd
Basic Peak	9.16c	30.4	162	0.16	NDd	5.6	94.2	10.0	90.0	NDd
Starting Material	8.7–9.1c	29.9	171	<0.01	19.8	68.7	11.6	0.2	99.7	0.2

a Acceptance criteria for test material: Concentration (30 ± 3 mg/mL); Osmolality (112–208 mOsm/kg); Endotoxin limits (<5 EU/kg which translates to <0.5 EU/mg of mAb based on the 10 mg/kg dose in rats).

b n, number of samples per test material.

c R², Coefficient of determination = 0.997.

d ND, not detected.

Table 3 Analytical characterization of isolated charge variant fractions

Method	Percentage of variants detecteda
Acidic Variant Fraction
IEC +/− Sialidase treatment	29% Sialylated
Reduced CE-SDS	7% Incompletely reduced
Non-reduced CE-SDS	29% Reduced disulfide
Boronate chromatography	17% Glycated
Peptide Map with Mass Spectrometry	18% Deamidatedb
Basic Variant Fraction
Peptide Map with Mass Spectrometry (for identification) and IEC (for quantification)	85% C-terminal heavy chain variants
	15% N-terminal Val-His-Ser light chain variants

a Representative values from isolated acidic and basic peak fractions are shown.

b Deamidated forms could not be quantified by UV absorbance (due to co-elution) or MS (due to differences in the ionization of deamidated and non-deamidated forms) and were therefore estimated based on the percentage of unaccounted acidic fraction forms.

Table 4 Comparison of all charge variant fractions to evaluate their kinetics of binding to rat FcRn, binding responses (in response unit) and their in vitro binding specific activity

Test material	Kinetic analysis and binding affinity to rat FcRn at pH 6.0 (n = 4)a				Response unit (RU) (n = 8)		% Specific activity (%CV)c
	ka1 (10^4 M^−1s^−1)	kd1 (10^−2 s^−1)	KD1 (10^−7 M)	Chi^2 (RU^2)	25 nM	250 nM
Acidic Peak	6.84 ± 1.28	4.57 ± 0.51	6.96 ± 2.09	0.259 ± 0.063	10.0 ± 1.3	32.3 ± 3.4	83 (10)
Main Peak	8.76 ± 0.83	4.67 ± 0.28	5.38 ± 0.76	0.214 ± 0.045	11.9 ± 1.3	35.7 ± 3.7	98 (17)
Basic Peak	8.18 ± 0.97	4.34 ± 0.39	5.40 ± 1.07	0.284 ± 0.037	11.9 ± 1.3	36.5 ± 4.0	95 (10)
Starting Materialb	7.99 ± 0.87	4.48 ± 0.22	5.67 ± 0.82	0.213 ± 0.042	11.9 ± 1.4	35.7 ± 4.0	100 (11)

a k_a1, first association rate constant; k_d1, first dissociation rate constant; K_D1, first dissociation equilibrium constant; Chi², a measure of the average squared residual (the difference between the experimental data and the fitted curve). All statistical analyses were performed using multiple regressions. The data shown are mean ± standard deviation.

b Binding of mAb starting material to rat FcRn is shown for reference only.

c In vitro binding potency analysis by inhibiting cell proliferation of all mAb fractions compared with reference material; CV, coefficient of variation of n = 3.

Table 5 Pharmacokinetic parameters of all mAb fractions in normal rats

Test material	Study (n = 12)a	AUC0–14^b (µg*day/mL)			Cmax (µg/mL)	Tmax (day)	Half-lifeb (day)
		Mean ± SD	Geometric mean	Geometric mean ratio (90% CI)c
Acidic Variant	IV	910 ± 73.1	907	0.963 (0.905, 1.03)	261 ± 27.3	NAd	8.09 ± 2.02
Main Peak	IV	895 ± 85.5	891	0.946 (0.884, 1.01)	256 ± 24.2	NAd	7.49 ± 1.90
Starting Material	IV	946 ± 94.6	942	NAd	245 ± 26.9	NAd	10.1 ± 0.942
Acidic Variant	SC	410 ± 37.2	408	0.992 (0.925, 1.063)	40 ± 3.2	4.3 ± 1.4	NAd
Main Peak	SC	399 ± 55.1	395	0.961 (0.872, 1.058)	39 ± 4.6	3.4 ± 1.2	NAd
Basic Variant	SC	391 ± 75.9	383	0.932 (0.811, 1.072)	39 ± 4.8	4.3 ± 1.8	NAd
Starting Material	SC	413 ± 29.1	411	NAd	38 ± 4.3	4.7 ± 1.8	NAd

a For the IV study the material was isolated with ion exchange chromatography and included the acidic fraction, main peak and starting material. Whereas, for the SC study the material was isolated by displacement chromatography and included the acidic and basic fractions, main peak and starting material; n, animals per group.

b Area under the curve (AUC) and half-life, were calculated for individual animals using non-compartmental analysis and are reported as the mean ± standard deviation of n = 12. Student's t-distribution was used to determine statistical comparison to starting material.

c CI, confidence interval.

d NA, not applicable.

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