The growing market for biopharmaceuticals1 brings new challenges for drug developers and manufacturers in assuring product quality and safety, and much of this can be attributed to product variability caused by post-translational modifications (PTMs).
The majority of biological drugs are proteins, and most proteins undergo some form of post-translational modification (PTMs) after being translated from an mRNA molecule. The most common PTMs include glycosylation, oxidation and deamidation, but there are many different types of PTM and they can occur at numerous sites within a protein affecting biological activity, half-life and immunogenicity.2 Characterizing PTM patterns is therefore a complex and time-consuming part of biopharmaceutical development and production.
PTMs in antibody-based drug developmentGiven the intense effort that is currently being invested in immunotherapy research, it is no surprise that understanding the effect of PTMs on antibody-based drugs is a research priority. PTMs, especially glycosylation, can have wide-ranging effects on potency and immunogenicity of antibodies – influencing their function and clearance.
“Different PTMs can affect different functional attributes of the drugs, depending on the mechanism of action,” says Dr John Schiel, Research Chemist at the US National Institute of Standards and Technology (NIST) who researches new methods for characterizing PTMs in antibodies. “Glycosylation of antibody drugs, for example, can influence their ability to become engulfed in cells and stored, thereby affecting the lifetime of the drug in the body,” explains Schiel. “There are also certain forms of glycans that excel at recruiting immune system cells to bind to cancer cells, and they can influence the Fc effector functions of the antibody drug itself, such as mediating antibody-dependent cellular cytotoxicity.”
A toolbox of around 30 different analytical assays is currently used to characterize biopharmaceuticals, says Schiel, and some of those inform on PTMs. “The current state of the art technique is to use liquid chromatography-mass spectrometry (LC-MS) peptide mapping, carried out during early drug development to dig deep into the product and understand what PTMs are there, how they might affect structure function, and start to get an idea of the levels of PTM.”
This information can be coupled with quality control (QC) assays performed later to give a global idea of PTMs in the sample, yet current QC assays don't tell you the exact location of the PTM. “A good example of this is with cation exchange chromatography,” says Schiel. “This generates a peak that is the main compound and side peaks that have PTMs. The side peaks all come out together and it's hard to tell which peak goes with which PTM.” The goal now is to adapt state-of-the-art peptide mapping techniques into the QC environment, a technique called the multi-attribute method (MAM).3 “Instead of setting specifications on that broad peak in cation exchange, we're trying to set parameters on specific peptides and have more “attribute specific” controls to routinely monitor how much PTM is actually present at a specific position in every lot of drug.”
Another important asset in the toolkit arising from NIST research is a standardized monoclonal antibody to support researchers developing antibody-based drugs. Issued in 2016, the NISTmAb is a biopharmaceutical-grade IgG1 monoclonal antibody. “We've characterized it comprehensively and made it publicly available,” explains Schiel. “It allows researchers to develop new analytical techniques or evaluate the robustness of their existing PTM characterisation methods using a single standard antibody.”
The impact of PTMs on biotherapeutic formulationOne of the key challenges is establishing how to mitigate changes in PTMs that occur over time during the early formulation of biotherapeutics, says Paul Dalby, Professor of Biochemical Engineering and Biotechnology at University College London. His lab is working on better ways to reliably predict the stability of protein-based drugs before clinical development is too advanced.
“The current method used in industry is to use “accelerated degradations” where you expose the drug to extremes – high temperatures, shaking, UV light – and look at the type of degradation that occurs and how fast it happens. You then extrapolate this to predict how the drug will perform on the shelf,” explained Dalby.
This might be enough to get a working formulation into your initial trial, but while the phase one study is ongoing, developers will also be incubating the formulation at its proposed storage temperature for the term of its shelf-life. This tells you whether your formulation was genuinely good or not. “Then you have a difficult decision to make,” says Dalby, “if your formulation wasn’t good enough, do you go back and reformulate – or do you set a shorter shelf-life for your product?” In some cases, it can be necessary to reformulate and complete bridging studies for a clinical trial, to show that the product will be OK once it’s on the market.
One of the problems with accelerated degradation studies, says Dalby, is that they don’t tend to correlate very well with shelf-life because the incubation at relatively high temperatures induces different protein modifications to those that occur at low temperatures. His group has developed new spectroscopic methods that are better at reliably predicting changes to the protein product.
“Let’s say that over a three-month period of incubation under high-stress conditions, your technique can pick up one in 100 proteins with PTMs. If you had a technique that could pick up one in 1000 proteins with PTMs then you wouldn’t need to incubate it under such extreme conditions. Our approach is to try and increase the sensitivity of detection of PTMs, so that you could detect changes at lower levels of stress that are more representative of the drug’s intended storage and use.”
A growing challenge in biopharmaceutical developmentAs biopharmaceuticals continue to offer great hope of treating previously intractable diseases, with the potential to personalise them to patients, finding new ways to characterize, control – or even predict – variation in their biological properties will be paramount. Mapping and measuring the levels of PTMs is already a crucial component of early biologic drug development but is by necessity now starting to move into QC processes too. New technologies that can easily characterize PTMs with high sensitivity will be an essential part of the drug developer’s toolkit.
- Mordor Intelligence, February 2019. Biopharmaceuticals Market - Growth, Trends, and Forecast (2019 - 2024). [Online] Available: https://www.mordorintelligence.com/industry-reports/global-biopharmaceuticals-market-industry
- Li W, Kerwin JL, Schiel J et al. (2015) Structural Elucidation of Post-Translational Modifications in Monoclonal Antibodies. In: State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NISTmAb Case Study. Chapter 3, pp 119–183. ACS Symposium Series, Vol. 1201 DOI: 10.1021/bk-2015-1201.ch003
- Rogers R, Abernathy M, Richardson D et al. View on the Importance of Multi-Attribute Method for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy. AAPS Journal 2018; 20: 7 DOI: 10.1208/s12248-017-0168-3