Antibody Production Cell Lines
Pablo Umana, Ph.D., CSO of GlycArt Biotechnology (www.glycart.com), will be discussing the use of GlycoMab® for engineering antibody production cell lines to produce therapeutic antibody glycoforms with modified glycosylation patterns. This modification is associated with higher biological activity and, hopefully, higher efficacy in the clinic.
Using CHO cells, the researchers add genes coding for an antibody along with a gene that encodes for a sugar-processing enzyme. “This works by over-expressing the enzyme (beta-1, 4-N-acetylglucosaminyltransferase-III) that causes production of bisected nonfucosylated sugars,” says Dr. Umana. Sugars normally produced by CHO cells are nonbisected fucosylated sugars. “Lack of this core fucose residue increases binding affinity to antibody receptors present on the immune system cells that recognize the antibody and are important for mediating the biological activity of the antibody,” he explains. Since the technology is the same used to develop antibodies, it’s easily applied.
It’s only been in the past few years that companies have begun to manipulate post-translational modifications, especially glycosylation, to create new forms of therapeutic proteins. “In the case of antibodies, alternative approaches to reduce the content of glycosylated sugars are a really unique example of how to modify the sugars and increase specific biological activity to make it more potent. We were the first to use recombinant DNA technology to manipulate and control the glycosylation profile of antibodies produced in mammalian cells to increase biological activity,” Dr. Omana states. The company currently has several antibodies modified using this technique in preclinical stages
Biacore (www.biacore.com) says that its partners and clients are increasingly using the company’s biosensors, based on real-time, label-free protein interaction analysis technology, in a process analytical setting. The goal is to assess the comparability of biological products between batches, or detect even slight protein alterations, such as post-translational modifications, following scale-up.
Biacore’s systems use a target immobilized on a sensor surface to reveal the specificity and rates of molecular binding interactions. The technology can analyze reaction rate kinetics (and thereby define affinity) and specificity, as well as concentration, points out Fredrik Sundberg Ph.D., the company’s director of global pharma operations.
“Using our technology, the interaction of an analyte with its binding partner, such as antibody to antigen or recombinant protein to target, is determined in terms of the rates of association and dissociation, presented as a graphical ‘sensorgram,’ the curve of which provides a unique ‘fingerprint,’” he notes.
“Any change in rate kinetics due, for example, to an increase in the off-rate—i.e., faster dissociation of the binding complex—will result in a change in the sensorgram fingerprint, even if the overall affinity of the protein determined by other kinds of end-point assay, remains the same. As a result, even minor modifications in the protein due, for example, to post-translational modifications or aggregation, will be detected, in real time. Once flagged, the nature of any modification to the protein can be identified using other technologies, such as size-exclusion HPLC, LC/MS and amino acid sequencing.”
Biacore believes this type of analysis could play an important role in all stages of biomanufacturing processes, as well as in batch release testing or lot-to-lot consistency testing of therapeutic protein batches.
“By quantifying the on-rate and off-rate constants for a particular batch, and setting an acceptable variable, it becomes fast and straightforward to compare data between process batches,” continues Dr. Sundberg. “A number of our major pharmaceutical and vaccine clients are now regularly using our technology in this area, and it is an application we will be actively promoting for our systems.”