shutterstock_556658050The onset of Biopharma has added tremendous value to the therapeutic portfolio. Peptide and protein-based therapeutics in the form of biotherapeutics/biopharmaceuticals/biologics have gained widespread popularity, as they are well accepted by our body and offer reduced side effects compared to the traditional small molecule drugs. In addition, these biopharmaceuticals are more targeted which  ensures higher efficacy. This wonderful combination of increased efficiency and fewer (and definitely fewer when compared to traditional small molecules) side effects deliver a knockout punch to a host of the most feared diseases — a dream dreamt by many is now a reality! However, as sweet as the results are, the journey to successfully designing, discovering and making biopharmaceuticals is not only complicated but is laced with layers of complexities. From the clone selection process to characterization of biologics within the human system; from ensuring their efficacy and ensuring product quality in each and every batch; from development to production — a lot of hard work, diligence, and perseverance is needed.

After characterizing a biopharmaceutical, the next steps comprise pharmacokinetic analyses leading to  targeted quantitation. For small molecules, these stages are primarily addressed by LC-MS techniques, but for large molecules like proteins, quantitative analysis is often addressed by ligand binding assays, and in recent times, LC-MS techniques. Owing to their increased specificity, selectivity, less vulnerability and high dynamic range, LC-MS techniques have been getting increased attention from bioanalytical scientists — the brains behind successful quantitative analysis of every single drug/biopharmaceutical. However, even if you feel that LC-MS is a superior technology or should be treated as the preferred technique, you still have to decide between a high resolution accurate mass spectrometer and a nominal mass spectrometer. When it comes to quantitative analysis of biopharmaceuticals in biological matrices, both offer some significant advantages; the first step is understanding what you want or need to achieve. The table below might help with your decision making.

Why High Resolution Accurate Mass?

Why QqQ?

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  • Confidence in confirmation
  • Confidence in operation, reliability
  • High resolution, accurate mass for non-target screening
  • Routine, targeted quantitation of samples in a high-throughput environment
  • Retrospective search of new compounds
  • Ensures high productivity in routine quantitation labs aiming for high selectivity & sensitivity
  • Sensitive, simultaneous non-targeted and targeted quantitation in a non-regulated environment
  • Addresses regulatory concerns and  ensures seamless productivity with established/customized methods

In the targeted world of quantitative analysis of pharmaceutical and biopharmaceuticals (DMPK, Phase I-III), a world that has been traditionally dominated by triple quadrupole MS, the impact of transitioning from small molecules to large molecules is felt in a big way. The biopharmaceuticals are significantly different from those of small molecules, not only in terms of size, but also in terms of their behavior. The biopharmaceuticals have multiple charge states, not just one – a typical feature found in small molecules. From a method development perspective, the selection of the right SRM may not depend on the most intense signal and one may need to pay more attention to fragmentation of the peptide from the N-/ C-terminal. When it comes to protein based molecules, perhaps the most important aspect to consider is the size of the molecules, which are significantly larger than what triple quadrupoles are capable of handling. Hence, one may need to cut the gigantic protein molecules (molecular weight: ~160 KDa) into smaller peptides via a digestive process. This process might sound like a combination of reasonable and relatively intuitive steps, but keep in mind that establishing and optimizing these processes can be complicated. Essentially, this is a huge stack of needles, from which you need to identify and quantify one particular type of needle. Not an easy task to say the least!

To address this challenge of developing a robust, sensitive, reliable LC-MS method to quantify biopharmaceuticals, a comprehensive solution is needed that comprises the following: sample digestion, clean-up and purification processes; an ideal liquid chromatography instrument that gives you the desired resolution, sensitivity, and speed; an optimal triple quadrupole MS platform that not only delivers high sensitivity, but also offers robustness, reliability and reproducibility; and last but not least, the software that guides you through the process.

How do you get started? When it comes to developing a robust, reliable, sensitive workflow for quantitation of biopharmaceuticals, Thermo Fisher Scientific offers some significant expertise and advantages to the users with both platform (LC-MS technology) and application solutions. Whether it is the use of a regular UHPLC with TSQ Endura for medium-high sensitivity, or a nanoLC with TSQ Quantiva for ultimate high sensitivity, there are several proof points available. The webinar from Dr. Eric Kuhn from Broad Institute (Cambridge, MA), Dr. Jun Qu (SUNY B, New York), and Dr. Nebiyu Ali-Abshiru (Northwestern University) demonstrate the power of a comprehensive workflow solutions in conducting biopharmaceutical quantitation. From sample treatment all the way through to the actual MS platform, the approach to address the challenges in the transition from small to large molecules must start by implementing an entire solution.

Dr. Eric Kuhn, Dr. Jun Qu, and Dr. Nebiyu Ali-Abshiru’s webinars also cover how workflows are the key large molecule characterization.