86 BIOTECHNOLOGY
Fig. 3. Off-target interactions. The frequency responses are obtained from antibody interactions to transfected (A) and not transfected (B) CHO cells. The specific antibody-receptor interaction is clearly observed in the dissociation phase (A, after 84s), while off-target interactions occurs while the antibody is continuously injected over the cells during the association phase (arrows).
dissociation rate, due to higher avidity and rebinding.
In the secondary binding assay, more of a secondary antibody binds to the aggregated proteins, which has more epitopes available for binding and thus
gives a higher shift in frequency response.
In contrast to the off-rate screening, the third assay points out the association phase, where aggregated proteins gain a higher frequency response when the surface is saturated, due to the higher loading of mass.
Receptor clustering Clustering of receptors and antigens is a natural event and affects binding properties such
as kinetic rate and affinity.3 Different protein products may interact differently to clustered receptors than non-clustered ones.1
In addition, the cell
membrane with its variety of components may lead to natural interactions between the protein product and other molecules than the target antigen.
Measuring the protein binding in a cell-based assay provides an added value to the kinetic information and is more biologically relevant than measuring the protein-antigen binding in a purified biochemical assay.
SWISSMADE
In this chapter, an antibody-cell interaction study is shown to illustrate off-target interactions. In the Attana Cell 200 biosensor, CHO cells and transfected CHO cells were used to distinguish specific and off-target interactions.
Te result clearly shows that the antibody interacts specifically to
REFERENCES: 1
the receptor in the transfected cells and also to other membrane components (shown as a fast interaction giving the typical shape of a hill, arrows in Fig. 3). Te data was fitted to a 1:2 binding model and the affinity calculated to 7nM (specific interaction) and 1.4µM (off-target interactions). It is important to detect and evaluate biological natural events at an early stage of the therapeutic protein product development.
Tis is to secure desired effects and limit unwanted immune responses against the products, both for patient safety and product efficacy.
Te label-free Attana Cell 200 biosensor can be used for studies of many of the factors mentioned in the FDA guidelines.1
For more information ✔ at
www.scientistlive.com/eurolab
Samuel Altun, Lena Heffler and Teodor Aastrup are with Attana, in Stockholm, Sweden.
www.attana.com
US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, & Center for Biologics Evaluation and Research. (2013, February). Immunogenicity Assessment for Terapeutic Protein Products. Guidance for Industry, pp. 1-32.
2
Zurdo, J, Michael, R, Rebecca, Y, Hedman, K, & Aastrup, T (2011, June). Improving the Developability. Innovations in Pharmaceutical Technology, pp. 34-40.
3
Aastrup, T (2013). Talking Sense. Innovations in Pharmaceutical Technology, pp. 48-51.
For more information ✔ at
www.scientistlive.com/eurolab
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92