Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity.
View Article and Find Full Text PDFMonoclonality of mammalian cell lines used for production of biologics is a regulatory expectation and one of the attributes assessed as part of a larger process to ensure consistent quality of the biologic. Historically, monoclonality has been demonstrated through statistics generated from limiting dilution cloning or through verified flow cytometry methods. A variety of new technologies are now on the market with the potential to offer more efficient and robust approaches to generating and documenting a clonal cell line.
View Article and Find Full Text PDFMethods Mol Biol
February 2012
A flow cytometry method using a nonfluorescent reporter protein was developed for rapid, early-stage identification of cells producing high levels of a recombinant protein of interest. A cell surface reporter protein is coexpressed with the protein of interest, and the reporter protein is detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the protein of interest are linked by an IRES so that they are transcribed in the same mRNA but are translated independently.
View Article and Find Full Text PDFHere we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest.
View Article and Find Full Text PDFFlow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently.
View Article and Find Full Text PDFLong-lasting synaptic plasticity and memory requires mRNA translation, yet little is known as to how this process is regulated. To explore the role that the translation repressor 4E-BP2 plays in hippocampal long-term potentiation (LTP) and learning and memory, we examined 4E-BP2 knock-out mice. Interestingly, genetic elimination of 4E-BP2 converted early-phase LTP to late-phase LTP (L-LTP) in the Schaffer collateral pathway, likely as a result of increased eIF4F complex formation and translation initiation.
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