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Biotherapeutics: What’s the Difference Between Template-Driven and Non-Template-Driven Features?

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Staying informed about what’s going on in the biopharmaceutical industry means knowing that biotherapeutics, or biologics, have become a main focus. Learning more about human genetics and disease has increased the number of potential targets for these highly specific compounds.1 Biotherapeutics are being used successfully to fight diseases that haven’t previously had effective therapies.2 The change in focus from small molecule pharmaceuticals to protein-based biotherapeutics does involve some new challenges, however, in understanding what determines the features of a biologic – and how to characterize them.

Small Molecule vs. Protein-Based Biotherapeutics
Small molecule pharmaceuticals are produced through chemical synthesis, sometimes with more than one possible pathway. Their final structure can be determined with a high level of certainty, using traditional analytical techniques.3 Protein-based biotherapeutics, on the other hand, are made by living systems, in situ, through recombinant DNA technology.4 The primary amino acid sequence of the biotherapeutic is the result of manipulating a cell into translating a specific genetic sequence to produce the desired compound.5

The features of the biotherapeutic depend on the accuracy of the translation and other process-specific conditions. The development process can result in three types of features within the biotherapeutic: template-driven features, non-template-driven features, and a combination of the two. Unwanted errors in each of these cases can impair the safety and effectiveness of the biologic.5

Template-Driven and Non-Template-Driven Features
The impact of an individual feature of the biotherapeutic is often hard to predict. The consideration of all the possible types of features can make that assessment even more difficult.5

Template-driven features are determined at the DNA level. They are a direct result of the primary amino acid sequence of the protein, established during translation. If an incorrect nucleotide has previously been incorporated into the DNA during replication, it can lead to a subsequent error in the primary sequence of the therapeutic.5

Non-template-driven features aren’t determined at the DNA level and can be influenced by a variety of things. They are non-specific, variable, and can be unpredictable. Glycosylation (O-linked, N-linked, and glycation) is an example of a  non-template-driven feature. The different attachment methods and locations for the sugar molecule can create a variety of glycosylation-related features.5

Some other features, like oxidation, can come from both template-driven and non-template-driven events. These kinds of modifications can occur after translation, but only after a specific sequence or amino acid is present. Oxidation mainly occurs on methionine, in the presence of reactive oxygen species. Deamidation and disulfide bond formation are other examples of combination features.5

Characterizing Biotherapeutics
Because protein-based biotherapeutics are much more complex than small molecule therapies, characterizing them can be equally complex. With the variety of possible template-driven and non-template-driven features, it can be difficult to determine the final structure with certainty.6 New and powerful analytical techniques are required to gain what information is available. Traditional methods are typically slow and overly complicated. Because definitive analysis of the end product is not always possible, monitoring the development pipeline for a biotherapeutic becomes critically important.6

There are some ways to look at the results of the complex combination of features and modifications. SCIEX provides innovative technology to increase the throughput of existing methods, and to simplify complex analyses using high resolution mass spectrometry and powerful software algorithms:

  • Charge heterogeneity analysis using capillary isoelectric focusing (cIEF) can provide a macro-level picture of the protein isoforms within a formulation without directly measuring the modifications. The cIEF technique identifies different protein isoforms based on their isoelectric point (pI) with a high level of resolution.5
  • Capillary electrophoresis can more directly measure modifications by looking at released glycans as a measure of the extent of glycosylation. CE analysis is helped by SCIEX kits and automation to overcome its throughput challenges.5 
  • High resolution mass spectrometry analysis, designed by SCIEX specifically for biologics, can provide confirmatory analysis of the primary amino acid sequence of the biotherapeutic.5

Read more about SCIEX solutions to analyze for template-driven and non-template-driven features here.


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