Anti-Ranibizumab Antibodies

Antibodies for bioanalytical assays for ranibizumab and biosimilar products

These inhibitory and non-inhibitory, recombinant monoclonal antibodies are highly specific for the monoclonal antibody Fab fragment ranibizumab (Lucentis) or the complex of ranibizumab with its target, vascular endothelial growth factor A (VEGF-A). Ranibizumab was created from the same parent mouse antibody as bevacizumab (Avastin) and some of our anti-ranibizumab antibodies also detect bevacizumab (Table 1).

Anti-Ranibizumab-VEGF Complex Specific Antibody

Anti-Ranibizumab-VEGF Complex Specific Antibody

This Type 3 monovalent Fab antibody recognizes the ranibizumab-VEGF complex; it does not bind to ranibizumab or VEGF-A alone. It can be used to develop a pharmacokinetic (PK) antigen capture assay, enabling detection of free drug.
Catalog Number: HCA304

Anti-Ranibizumab Non-Inhibitory Antibodies

Anti-Ranibizumab Non-Inhibitory Antibodies

These two non-inhibitory Type 2 antibodies do not inhibit the binding of ranibizumab to VEGF-A and can be used in a PK antigen capture assay, enabling detection of free drug.
Catalog Numbers: HCA306, HCA307

 

Anti-Ranibizumab Inhibitory Antibodies

These two Type 1 antibodies inhibit the binding of ranibizumab to its target VEGF-A, and therefore detect free drug. The antibodies are in fully human IgG1 format and suitable as a surrogate positive control or calibration standard in an anti-drug antibody (ADA) assay.
Catalog Numbers: HCA305, HCA185

These recombinant, monoclonal antibodies are generated using Human Combinatorial Antibody Library (HuCAL®) technology and CysDisplay®, a proprietary method of phage display, and employing guided selection methods to obtain highly targeted reagents. The result is highly specific and high affinity antibodies, ideal for development of PK assays. HuCAL antibodies are fully human, and when in full length IgG format they can be used as a control or calibrator in an ADA assay. The in vitro production of recombinant antibodies results in a consistent, secure supply of these critical reagents throughout preclinical development and clinical trials.

More information about anti-idiotypic antibody binding types 1, 2 and 3 and their properties

Anti-biotherapeutic antibody quality control and characterization

Table 1. Antibodies Specific to Ranibizumab

Catalog #

Clone

Antibody Specificity

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

HCA304

AbD29928

Ranibizumab
/VEGF complex &

Bevacizumab/VEGF complex3

Complex-specific

Fab-FH1

1

PK antigen capture assay

HCA306

AbD29858

Ranibizumab & Ranibizumab/VEGF complex

Non-inhibitory

Fab-FH1

18

PK antigen capture assay

HCA307

AbD29865

Ranibizumab & Ranibizumab/VEGF complex

Bevacizumab & Bevacizumab/VEGF complex3

Non-inhibitory

Fab-FH1

5

PK antigen capture assay

HCA305

AbD16746_hIgG1

Ranibizumab & Bevacizumab3

Inhibitory

Human IgG1

22

ADA assay

HCA185

AbD17976_hIgG1

Ranibizumab & Bevacizumab3

Inhibitory

Human IgG1

0.42

ADA assay

1 F=DYKDDDDK-tag H=His-6-tag
2 Affinity measured in the monovalent Fab format by real time,
label free molecular interaction analysis on immobilized bevacizumab
​3 Ranibizumab originates from the same parental mouse antibody as bevacizumab; it has four amino acid exchanges in the heavy chain and one amino acid exchange in the light chain compared to bevacizumab

Related Products

Ranibizumab drug target Recombinant Human VEGF-A (PHP293)


Pharmacokinetic Assay

Schematic image of PK Bridging ELISA. Capture antibody, Fab format (purple), fusion protein drug (gold-green), detection antibody, Ig format (blue), labeled with HRP

Schematic image of PK antigen capture ELISA with Type 3 antibody. Antigen/drug target (red), Fab fragment antibody drug (gold), Type 3 drug-target complex specific antibody, Fab format (purple).

Fig. 1. Ranibizumab PK antigen capture ELISA using antibody HCA304.

Fig. 1. Ranibizumab PK antigen capture ELISA using antibody HCA304.

 


Measurement of free drug with Human Anti-Ranibizumab Antibodies

In Figure 1, a microtiter plate was coated overnight with Human Recombinant VEGF-A (PHP293) at a concentration of 5 µg/ml. Washing and blocking was performed with PBST + 5% BSA. PBST with 10% human serum spiked with increasing concentrations of ranibizumab was added. Detection was performed using Human Anti-Ranibizumab/VEGF Antibody, clone AbD29928 (HCA304) at a concentration of 2 µg/ml, followed by an HRP conjugated anti-DYKDDDDK tag antibody in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Schematic image of PK Bridging ELISA. Capture antibody, Fab format (purple), fusion protein drug (gold-green), detection antibody, Ig format (blue), labeled with HRP

Schematic image of PK antigen capture ELISA with Type 2 antibody. Antigen/drug target (red), Fab fragment antibody drug (gold), Type 2 non-inhibitory antibody, Fab format (purple).

Fig. 2. Ranibizumab PK antigen capture ELISA using antibody HCA307.

Fig. 2. Ranibizumab PK antigen capture ELISA using antibody HCA307.


In Figure 2, a microtiter plate was coated overnight with Human Recombinant VEGF-A (PHP293) at a concentration of 5 µg/ml. After washing and blocking with PBST + 5% BSA, 10% human serum was added spiked with increasing concentrations of ranibizumab. Detection was performed using Human Anti-Ranibizumab Antibody, clone AbD29865 (HCA307) at a concentration of 2 µg/ml, followed by an HRP conjugated anti-DYKDDDDK tag antibody in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: Ranibizumab PK antigen capture ELISA protocol HCA304, HCA306, HCA307


Anti-Drug Antibody Assay – Bridging ELISA

Schematic image of PK Bridging ELISA. Capture antibody, Fab format (purple), fusion protein drug (gold-green), detection antibody, Ig format (blue), labeled with HRP

Schematic image of ADA bridging assay. Monoclonal Fab fragment antibody drug as capture antibody and detection antibody labeled with HRP (gold), fully human anti-idiotypic antibody, Ig format (blue).

Fig. 3. ADA bridging ELISA using antibody HCA305

Fig. 3. ADA bridging ELISA using antibody HCA305

 

Immunogenicity assay using Human Anti-Ranibizumab Antibody as calibration standard

In Figure 3, ranibizumab was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with PBST + 5% BSA, PBST with 10% human serum was added, spiked with increasing concentrations of Human Anti-Ranibizumab Antibody, clone AbD16746_hIgG1 (HCA305). Detection was performed using HRP conjugated ranibizumab at 2 µg/ml in HISPEC Assay Diluent, and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements. HRP conjugation of ranibizumab was performed using a LYNX Rapid HRP Antibody Conjugation Kit®.

Protocol: Ranibizumab ADA bridging ELISA protocol


Specificity of antibodies to Ranibizumab, Ranibizumab/VEGF complex and Bevacizumab

In Figures 4, 5 and 6, a microtiter plate was coated overnight with various antigens and drug-target complexes at a concentration of 5 µg/ml. After washing and blocking with PBST + 5% BSA, detection was performed using an anti-ranibizumab antibody at a concentration of 2 μg/ml followed by an HRP conjugated anti-DYKDDDDK tag antibody in HISPEC Assay Diluent (BUF049A), and QuantaBlu Fluorogenic Peroxidase Substrate.

Fig. 4. Specificity of Human Anti-Ranibizumab/VEGF Antibody HC304.
Fig. 4. Specificity of Human Anti-Ranibizumab/VEGF Antibody HC304.

HCA304 is a drug-target complex specific antibody that recognizes the ranibizumab/VEGF complex. It also detects the bevacizumab/VEGF complex (data not shown).

Fig. 5. Specificity of Human Anti-Ranibizumab Antibody HC306.
Fig. 5. Specificity of Human Anti-Ranibizumab Antibody HC306.

HCA306 is a non-inhibitory anti-idiotypic antibody, specific to ranibizumab, that does not cross-react with bevacizumab or bevacizumab/VEGF complex.

Fig. 6. Specificity of Human Anti-Ranibizumab Antibody HC307.
Fig. 6. Specificity of Human Anti-Ranibizumab Antibody HC307.

HCA307 is a non-inhibitory anti-idiotypic antibody, specific to ranibizumab that also recognizes bevacizumab and bevacizumab/VEGF complex.


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