Overcoming Challenges When Scaling Antibody Production

Tradeoffs are a constant source of challenge in any research lab. To get faster results, you will probably need to use more resources (people, money, supplies). The powerful lasers used to do live cell imaging may well kill those cells in the process. Purifying DNA often leaves you to choose between purity and yield.

Robot performing autosamplingWorking with biologics also involves a delicate balancing act. Producing compounds in biological models rather than by chemical synthesis offers many advantages, but it is not without certain challenges. One of those tradeoffs results from scaling up; the more plasmid that is produced, the greater probability of endotoxin contamination.

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Shooting for the Moon: Better Assays to Hit Our Cancer Research Targets

3239CA02_1AIn his address to the clinicians, researchers, and patients at the American Association for Cancer Research meeting in April, US Vice President Joe Biden, revealed that the goal of the #cancermoonshot initiative is to accomplish 10 years of cancer research in just five years, effectively doubling the pace of cancer research (1).

Treatments developed from cancer research have come a long way with dramatic differences in the experiences and prognoses for patients, just looking back over the last 25 years. How can we double the pace of cancer research? The #cancermoonshot will one, encourage data sharing among researchers, particularly data from clinical trials. Second, it seeks to increase collaboration across industry, academic and government scientists—each community being positioned to make unique contributions to the field. And third, the initiative looks to change the current grants award process that encourages scientists to keep data and results “quiet” until they can be published or protected legally as intellectual property.

Immunotherapy is an especially hot field in cancer research (2) that relies on the immune system to better fight cancer. Continue reading

Optimizing Antibody Internalization Assays: pHAb Dyes

22255190_pHabAmine_3D_image_050615-final-largePromega has recently developed a method that allows antibodies to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When an antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane, the antibody-dye-antigen complex is not fluorescent, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops, and the dye becomes fluorescent.

To demonstrate the broad utility of the pHAb dye for receptor mediated antibody internalization, two therapeutic antibodies, trastuzumab and cetuximab,which bind to HER2 and EGFR respectively, were selected for a case study (1). Both the antibodies, which are known to internalize were labeled with pHAb dyes using amine or thiol chemistry.

Parameters such as the impact of dye–to-antibody ratio on the antigen–antibody binding, change in fluorescence as a function of pH of free dye and labeled dye, and labeled antibody internalization as a function of pHAb conjugated antibody concentration were evaluated.

The results indicate that pHAb dyes are pH sensitive fluorescent dyes that enable the study of receptor-mediated antibody internalization.Internalization assays can be performed in a plate-based homogeneous format and allow endpoint assays as well as real-time monitoring of internalization. They further show that internalization can be monitored even at a very low amount of antibody which is very important during the early monoclonal antibody development phase when the amount of sample is limited and the antibody concentration in the samples is low. a complimentary approach, they  also showed that a secondary antibody labeled with pHAb dye can be used instead of labeling primary antibodies.

Literature cited

Nath, N. et al. (2016) Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye J.  Immunol. Methods epub ahead of print

Characterizing Immune-Modulating Antibodies Using Bioluminescence

Immune checkpoint pathways such as PD-1/PD-L1 and CTLA-4 are promising new immunotherapy targets for the treatment of cancer and autoimmunity. Immune checkpoint reporter-based bioassays provide a simple, consistent, and reliable cell-based assay to measure Ab function throughout the drug development pipeline.

The brief chalk talk below describes the assay principals of the reporter-based bioassay that monitors the functional blockade of PD-1/PD-L1 interactions.