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Feb 1, 2013 (Vol. 33, No. 3)

Challenging the Limits of Protein Characterization

  • Where Is It?

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    Researchers at Novo Nordisk use Orbitrap instruments for LC-MS/MS characterization of chemical degradation products of therapeutic peptides.

    Even without the help of pump parts and rubber curing agents, proteins and peptides can degrade over time. It’s important to know how and where such degradation takes place, so as to try to minimize the impact on the formulations. Beyond the chance of adverse immunological reactions, the biological activity may be compromised as well, for example, as a deaminated or cross-linked therapeutic loses its ability to engage with a receptor.

    Peter Kresten Nielsen, scientific director, protein chemistry at Novo Nordisk, examines the chemical stability of prototype formulations by incubating them at high temperatures and analyzing the degradation sites using a top-down mass spectrometry approach.

    “We have to provide feedback to the chemists who then synthesize the optimized peptide based on our MS input. We don’t have time to purify the degradation products, and collect fractions, and digest the peptides by protease, and so on, so we can save time and give fast input to further peptide design,” he said.

    Among the challenges he faces are detecting sites of isomerization of individual amino acids—for example, the conversion of aspartic acid (Asp) to isoAsp. “It’s really difficult to detect because there is no change in mass, so you can’t detect it by just normal MS fragmentation methods,” he lamented.

    Because the isomerization reaction changes the amino acid’s backbone, using specialized methods such as electron-transfer dissociation (ETD) to fragment the peptide will yield reporter ions that are unique to isoAsp. The problem is that the peak intensities for these ions are significantly lower than those for the common fragmentation ions, and can be indistinguishable from spurious noise.

    This is most often solved by first digesting with a protease that will cleave Asp peptides but not isoAsp—not an option for a top-down approach. “We’re working a lot on how to optimize this.”

    Similarly, Nielsen is faced with racemized peptides (L- and D- forms), which also have the same mass, but do not yield unique reporter ions. He has been using reference peptides that contain these modifications, and experimenting with MS combined with a combination of liquid chromatography and ion mobility spectrometry. “It looks promising,” he said, “but still it requires some optimization.”

  • Waiting to Inhale

    Once the drug is identified, it still has to be administered somehow, whether it’s sub-cutaneously, intramuscularly, intravenously, or perhaps orally or as a suppository. And there should be a reason behind the choice of delivery route. “Don’t do it just because you can—you really need to look at what is the unmet medical need, and then develop a target product profile,” said David Cipolla, senior director, pharmaceutical sciences, at Aradigm, which develops products for inhalation including macromolecular drugs.

    “You need to make sure that the rationale for the product makes sense after considering all the stakeholders, to look at the competition in the marketplace that you’re competing with, and to keep your product profile updated as development progresses,” he added.

    Cipolla discussed three principal scenarios for (re)positioning a drug for inhaled delivery. The first and most obvious is to treat lung-related disorders like asthma or lung cancer. The second is to find a lung-related indication for an existing drug, like using cyclosporine to prevent transplantation rejection. And the third is to use the inhalation route to treat a systemic disorder, to alter the pharmacokinetics (for pain relief, for example) or pharmacodynamics, or to avoid injection-related issues.

    There are a variety of inhalation delivery systems to choose from, each with its own advantages and disadvantages that need to be weighed. Dry powder inhalers and metered dose inhalers are small, and quick and easy to use, for example. A nebulizer is bulky, inefficient, and requires long dosing times, but it’s a well-established technology that can be used off-the-shelf. Other, more advanced systems can assure more efficient and accurate delivery, with comparable or better performance for the latter versus injections.

    Since biopharmaceuticals are manufactured in aqueous-based fermentation or cell culture systems anyway, Cipolla recommends going into initial safety and small proof of concept human studies using a standard jet nebulizer with a simple aqueous formulation.

    “And then in parallel you can develop, if needed, another formulation that will give you the commercial specifications, such as the dose and long-term stability. And once you know that it works, that’s when you spend the time and money on whatever you want as a final delivery configuration.”

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