BACKGROUND
Biologics have been developed from naturally occurring systems such as humans, animals, or microorganisms and include a wide range of products such as vaccines, blood and blood components, allergens, genes, tissues, and recombinant proteins. In contrast to most drugs that are chemically synthesized, most biologics are complex molecules that are not easily identified or characterized. Biologics have revolutionized the prevention, diagnosis, and treatment of cancer, autoimmune conditions, and several other diseases. However, biologics are much more expensive than chemically synthesized drugs due to their complex manufacturing process. Because of their high cost, there is a great demand to manufacture generics of biologics. These biosimilars are biological products that are highly similar to and have no clinically meaningful differences from an approved reference product.
The top 10 bestselling biologics in 2023 were proteins, including six monoclonal antibodies (mAbs) (Adalimumab, rituximab, trastuzumab, bevacizumab, infliximab, ranibizumab) and four other proteins (Etanercept, insulin glargine, pegfilgrastim, interferon beta-1α) with global annual sales more than $1.5 billion.
The therapeutic proteins require extensive analytical characterization to ensure safety, purity, and potency of the product. Liquid chromatography coupled to mass spectrometry (LC-MS) plays a major role in the analytical characterization of therapeutic proteins. LC-MS is particularly important to assess the amino acid sequence, molecular masses, posttranslational modifications (PTMs) (e.g., oxidation, deamidation, glycosylation), disulfide bonds, N- and C-terminal truncations, sequence variants, and host cell protein (HCP) impurities.
Figure 1: Intact mass and peptide mapping to analyze monoclonal antibodies (mAbs). After reductive alkylation (TCEP/IAA), enzymatic digestion with trypsin is commonly used to generate a peptide map. To ensure 100% sequence coverage, another experiment using a second enzyme might be needed. The peptide map can be used to confirm the amino acid sequence as well as to find posttranslational modifications and sequence variants.
MASTER PROJECT
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Improved monoclonal antibody characterization
The full analysis of mAbs by LC-MS is challenging and requires additional methods to the current repertoire for improving confirmed sequence coverages, sequence variants, and posttranslational modifications (PTMs).
For improved mAbs characterization, different enzymatic approaches in combination with LC-MS will be evaluated.
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Improved host cell protein (HCP) impurity analysis
HCPs derive from the biotechnological manufacturing process of the therapeutic proteins. Notably, chinese hamster ovarian (CHO) cells are commonly used to produce therapeutic proteins. HCPs can contaminate the product and can reduce the effectiveness of the drug or cause side effects in patients. Therefore, it is crucial to identify thoroughly HCPs. Commonly, they are detected by ELISA and LC-MS. ELISA is used to quantify known HCPs in high throughput, while LC-MS is able to identify and quantify HCPs without prior knowledge of the identity of the proteins.
Different approaches based on e.g., magnetic beads, filtration devices, precipitation methods, will be evaluated to increase the number of HCP identifications in mAbs solutions.
Abbreviations:
CHO Chinese hamster ovarian
HCP Host cell proteins
LC-MS Liquid chromatography-mass spectrometry
mAb Monoclonal antibody
PTM Posttranslational modification
Contact:
Main supervisor: Bernd Thiede, BMB, IBV
bernd.thiede@ibv.uio.no