Comparison of immunogenicity test methods used in clinical studies of infliximab and its biosimilar (CT-P13)

Figures & data

Figure 1. Schematic of methods to detect anti-drug antibodies. (A) In bridging ECL, samples are acidified then neutralized with ruthenylated RMP and biotinylated RMP. During incubation, ADAs are bound to both sulfo-tagged and biotinylated RMP to form an antibody–complex bridge, and samples are then loaded onto a streptavidin-coated electrode plate. After washing, only samples that contain ADAs bound to both sulfo-tagged and biotinylated RMP generate an ECL signal. (B) A bridging ELISA is based on the double-antigen format. RMP is used both during the solid-phase to capture antibodies against RMP and during the biotinylated detection phase with neutravidin–horseradish peroxidase. (C) In RIA, any radioactive complex extracted by anti-human lambda light chain antibodies is presumed to be RMP bound to ADA. This is because RMP is an antibody constructed solely of kappa light chains. (D) In HMSA, samples are incubated with florescent-labeled RMP. Resulting immune complexes are separated from the free label by SE-HPLC and the amount of ADA in the samples calculated from the resolved peak areas.

Table 1. An overview of the advantages and disadvantages of ECL, ELISA, RIA, and HMSA methods.

Assay	ECL [6,8,18]	ELISA [6,8,20]	RIA [23]	HMSA [17]
Summary of method	Better free drug tolerance than ELISA High serum tolerance Detection of low-affinity ADAs Better sensitivity than ELISA Hook effect may occur (false positives)	Washing may remove low-affinity ADAs Reduced detection of low-affinity ADAs when the bridging format is used False positives caused by non-specific binding of immunoglobulins or rheumatoid factors	Avoids denaturing effect of coating seen with other methods Can avoid the risk of false positives seen with ELISA Method is complex Requires long incubation time Safety of radioactive substances in the laboratory	Better sensitivity than ELISA Better free drug tolerance than ELISA

ADAs: Anti-drug antibodies; ECL: Electrochemiluminescent; HMSA: Homogeneous mobility shift assay; RIA: Radioimmunoassay.

Table 2. Comparison of sensitivity and drug tolerance level among the different assay methods.

Platform	Sensitivity/cut point	Drug tolerance
ECL	50.8 ng/ml sensitivity Floating cut point (similar to sensitivity level)	150 ng/ml ADAs to infliximab can be measured in the presence of >5 μg/ml RMP in serum
ELISA [19]	^†1.69 μg/ml cut point	^†1.40 μg/ml
RIA [24,36]	120 ng/ml (12 AU/ml) cut point	>2 μg/ml
HMSA [17]	0.56 μg/ml LLOQ 1.19 μg/ml cut point	36 ng/ml ADAs to infliximab can be measured in the presence of 60 μg/ml RMP in serum

^†These values cannot be conclusively determined, because infliximab interferes with the ELISA method.

ADAs: Anti-drug antibodies; AU: Arbitrary unit; ECL: Electrochemiluminescent; HMSA: Homogeneous mobility shift assay; LLOQ: Lower limit of quantification; RIA: Radioimmunoassay; RMP: Reference medicinal product.

Table 3. Historical ADA rates reported with CT-P13 or infliximab RMP using ECL, ELISA, and RIA assay methods^†.

Study (year)	Indication	Dose (mg/kg)	Concomitant therapy	ADA rate at primary end point % (n/N)^‡	Assay method	Ref.
Braun et al. (2008)	AS	5	NS	RMP, 6 (5/83) (week 102)	ELISA	[37]
Xu et al. (2008)		5 or 7.5	–	RMP, 8 (23/274)	ELISA	[38]
Ducourau et al. (2011)		5	MTX –	RMP, 0 (0/25) RMP, 21 (14/66)	ELISA	[27]
PLANETAS		5	–	RMP, 23 CT-P13, 27 (week 30)	ECL	[12]
Maini et al. (1998)	RA	3	MTX	RMP, 17 (week 26)	ELISA	[28]
St Clair et al. (2004)	RA	3	MTX	RMP, 14.5 (46/317)	ELISA	[31]
Bendtzen et al. (2006)	RA	3	MTX –	RMP, 40 (month 6) RMP, 50 (month 6)	RIA	[24]
Wolbink et al. (2006)	RA	3	MTX	RMP, 43 (22/51)	RIA	[25]
Rahman et al. (2007)	RA	3	–	RMP, 19.5	ELISA	[30]
Remicade^® SmPC (2009)	RA	3	–	RMP, 24	ELISA	[40]
Vultaggio et al. (2010)	RA	NS	MTX	RMP, 14.1(10/71)	ELISA	[32]
Ruperto et al. (2010)	Juvenile RA	3–6	MTX	RMP, 37 (26/71)	ELISA	[41]
Kosmač et al. (2011)	Juvenile idiopathic arthritis	3–5	MTX	RMP, 43 (9/21)	ELISA	[33]
Ducourau et al. (2011)	RA	3	–	RMP, 50 (3/6)	ELISA	[27]
Pascual-Salcedo et al. (2011)	RA	3	MTX –	RMP, 32 RMP, 37	ELISA	[29]
PLANETRA	RA	3	MTX MTX	RMP, 48 CT-P13, 48 (week 30)	ECL	[13]

^†The HMSA method has not been used to assess the immunogenicity of RMP in clinical studies of patients with rheumatic diseases, and so data are not summarized for this assay.

^‡Because the timing of serum collection can influence the results of ADA measurement, timing of measurements is presented, if available.

ADA: Anti-drug antibodies; AS: Ankylosing spondylitis; ECL: Electrochemiluminescent; HMSA: Homogeneous mobility shift assay; MTX: Methotrexate; NS: Not specified; PLANETAS: Programme evaLuating the autoimmune disease iNvEstigational drug CT-P13 in AS patients; PLANETRA: Programme evaLuating the Autoimmune disease iNvEstigational drug CT-P13 in RA patients; RA: Rheumatoid arthritis; RIA: Radioimmunoassay; RMP: Reference medicinal product; SmPC: Summary of product characteristics.

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