A therapeutic antibody’s efficacy depends not only on the Fab fragment and its binding activity to the target antigen, but also on the Fc fragment how it interacts with key Fc receptors. The half-life of the anti-body is predicted by the binding affinity of the Fc fragment towards FcRn (FCGRT&B2M), while the binding affinity between the Fc fragment and FcγRIIIa (CD16a) would influence the antibody’s ability to elicit ADCC (antibody dependent cell mediated cytotoxicity).
Potential candidates must therefore be tested against a panel of receptors during antibody engineering. ACROBiosystems offers a comprehensive collection of recombinant Fc receptor proteins, as well as their common variants, to help expedite your mAb development.
Pharmaceutical studies include a high purity requirement of their starting materials, so their production team performs both SDS-PAGE and HPLC analyses to the Fc receptors for QC purposes. An item of note is that it’s essential to use monomeric Fc receptor, as this represents its natural state.
If oligomers are allowed to form during production, the resulting avidity effect may result in an artificially enhanced binding interaction. ACROBiosystems has established strict internal control standards of monomer purity by HPLC testing to avoid this scenario, and only those batches meeting all purity requirements are released.
Figure 1. The purity of Human Fc gamma RIIA / CD32a (R167) Protein (Cat. No. CDA-H5221) is greater than 95% as determined in a HPLC analysis.
Bioactivity validated by SPR
There are numerous methods of determining the binding affinity between two molecules. However, the studies of Fc interaction are often done by SPR, as traditional ELISA is not appropriate for low affinity receptors such as CD16 and CD32. To assure the Fc receptors produced by ACROBiosystems deliver expected performance, the QC team uses the Biacore platform to test products against reference antibodies.
Figure 2. Immobilized Human Fc gamma RI / CD64, His Tag (Cat. No. FCA-H52H2) on CM5 Chip via anti-His antibody, can bind Herceptin with an affinity constant of 5.45 nM as determined in a SPR assay (Biacore T200).
Figure 3. Immobilized Human FcRn / FCGRT & B2M Heterodimer Protein (Cat. No. FCM-H5286) on CM5 Chip via anti-His antibody, can bind Herceptin with an affinity constant of 0.489 μM as determined in a SPR assay (Biacore T200).
High Batch-to-batch Consistency
To ensure consistent performance of the product, ACROBiosystems routinely applies rigorous quality control measures. The figure below shows that batch variation among the tested samples is negligible.
The performance of different batches of CD32b (CDB-H5228) in the same assay was compared, and the software analysis showed a very high similarity score, meaning high batch-to-batch consistency.
Figure 4. Immobilized Human Fc gamma RIIB / CD32b Protein (Cat. No. CDB-H5228) on CM5 Chip via anti-His antibody, can bind Rituximab with an affinity constant of 10 μM as determined in a SPR assay (Biacore T200).
Figure 5. Batch consistency of Human Fc gamma RIIB / CD32b (Cat. No. CDB-H5228). The Similarity for different batchs is more than 90%.
Biotinylated or Other Species Fc Receptors Available
Assay development can be made much easier through the use of biotin labeling. ACROBiosystems offers a variety of ready-to-use biotinylated Fc receptors. These proteins are produced using in-house developed labeling techniques, which confers high bioactivity and minimal batch-to-batch variation.
As well as this, the company has developed a series of Fc receptors of other species, which are ideal for the screening of non-humanized antibodies or the species cross reaction.
Figure 6. Biotinylated Human CD16a (V176), His Tag, Avi Tag (Cat. No.CDA-H82E9) captured on Biotin CAP- Series S Sensor Chip can bind Rituximab with an affinity constant of 0.261 μM as determined in a SPR assay (Biacore T200).
Figure 7. Immobilized Cynomolgus / Rhesus macaque FcRn Protein (Cat. No. FCM-C5284) on CM5 Chip via anti-His antibody, can bind Herceptin with an affinity constant of 0.403 μM as determined in a SPR assay (Biacore T200).
ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.
Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.
We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.
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