Article
Large molecule therapeutics, also known as biologics, biopharmaceuticals or biotherapeutics are, as the name suggests, large proteins or peptides that have a therapeutic effect. Large molecule therapeutics are generally manufactured through recombinant DNA technology, in living cell lines, and as such have complex structures. This means that they are less stable than their small molecule counterparts, and due to this, they are generally administered intravenously or intramuscularly. Large molecule therapeutics make up a huge portion of the wider pharmaceutical market, taking up 8 spots out of the top 20 best-selling drugs in 2022, with projected growth estimating that by 2025 the market will be worth $300 billion. Monoclonal antibodies have been the mainstay of biologics for over 30 years and technological advancement has resulted in the generation of multiple novel therapeutic molecules derived from their structure.
Ligand binding assays (LBA), also known as immunoassays, rely on the binding of ligand molecules to receptors, antibodies and other large proteins to detect a target analyte. In bioanalysis, ligand binding assays enable the quantification of therapeutic proteins/drugs & biomarkers alongside qualitative analysis of anti-drug antibodies (ADA) and neutralizing anti-drug antibodies (NAb) in biological samples. This is in addition to a multitude of diagnostic and medical uses in a wide range of other fields. First described in the 1970s, advancements in recombinant protein/antibody generation and detection systems have kept ligand binding assays at the forefront of large molecule bioanalysis. Ligand binding assays are most commonly performed as plate-based assays, although other technologies and platforms are becoming more widespread. The main detection strategies are chromogenic (colourmetric), fluorescent and chemiluminescent which all involve secondary reagents/antibodies conjugated with an enzyme or other reactive molecules. These are commonly visualised via the addition of a substrate solution allowing the detection of the target analyte.
At ACM Global Laboratories, our bioanalytical services and state-of-the-art Molecular Devices SpectraMax and Meso Scale Discovery (MSD) Quickplex plate readers enable colourimetric (ELISA method) and electrochemiluminescence/ECL (MSD) assays to be performed. Colourmetric assays are a cost-effective way to perform quantitative analysis whilst MSD assays provide a higher sensitivity/specific alternative with the addition of providing multiplexing capabilities.
Our large molecule bioanalysis service line provides a custom ligand binding assay development and validation service designed specifically for the investigation medicinal product (IMP) of interest. This enables the regulated bioanalysis of pharmacokinetic (PK), toxicokinetic (TK), ADA/NAb immunogenicity, and pharmacodynamic (PD) biomarker samples.
TK and PK research data is vital for the assessment of the absorption, distribution, metabolism and excretion of a target IMP from the body. PK/TK data for IMP is collected and evaluated routinely in all pre-clinical and clinical trials. This is because it is critical in assessing safety and efficacy as well as determining dose regimes and monitoring the toxicity of an experimental treatment.
Unfortunately due to their nature, large molecule therapeutics have a problem with potential immunogenicity, which means they can trigger an unwanted immune response. Therefore, a critical part of the phases of drug development is measuring the tendency of an IMP to trigger the generation of ADA and subsequently neutralising antibodies. Regulators expect immunogenicity testing for therapeutic proteins as well as other relevant classes of drugs where potential immunogenicity is predicted or established. The MSD bridging assay format has become the gold standard for ADA testing within the industry due to its specificity and sensitivity capabilities. ACM offers a comprehensive immunogenicity testing service including the reagent conjugation capabilities to facilitate the required testing.
Biomarker testing data is a great tool in drug development, depending on the “context of use” requirements, biomarkers can be useful in the early stages of development, helping to establish the early proof of concept of a therapeutic or in late-stage development being used as pharmacodynamic primary or secondary end-points in clinical trials, indicating the pharmacological effect of a drug. Ligand binding assays can support the full spectrum of biomarker analysis from large multiplexing exploratory panels used for wide-ranging non-regulatory evaluations to super-sensitivity custom-built single-plex assays, suitable to support investigating new drug submissions. ACM Global Laboratories can support the analysis of over 500+ biomarkers in clinical and pre-clinical species, from an exploratory level up to bespoke validated assays upon request.
Monoclonal antibody treatments such as Trastuzumab (trade name: Herceptin), Adalimumab (trade name: Humira) and the more recent Pembrolizumab (trade name Keytruda) have altered the approach to oncology and inflammatory treatments alongside a host of other therapeutic areas. Continued scientific progress has built on this established technology to enable the generation of bi-specific antibodies (BsAbs). Instead of binding to one target/antigen as traditional antibodies do, BsAbs can simultaneously bind to two different antigens or two different epitopes of the same antigen, enabling advanced therapeutic uses. There are currently 9 FDA approved bi-specific antibodies, mainly in the oncology space such as Amivantamab (trade name Rybrevant) but they are also used in other therapeutic areas such as Emicizumab (trade name Hemlibra) used to treat Hemophilia A.
Bi-specific antibodies are a type of fusion protein which have been enabled by rapid advances in recombinant DNA protein engineering. Fusion proteins cover a range of molecule types including chimeric and bi/tri-specific antibodies. The advantages of fusion proteins mean they can be tailored to the specific therapeutic of interest, for instance, adding an Fc domain taken from a monoclonal antibody to dramatically increase the serum half-life, or engineering animal antibodies to humanise them, thus reducing their immunogenicity. Etanercept (Enbrel) was the first fusion protein approved for the treatment of autoimmune diseases in 1998, which is comprised of two TNF receptors linked to an Fc portion of an IgG1. The market for Fc-fusion proteins was estimated to be worth approximately $27 billion in 2020 and is expected to rise to ~$89 billion by 2032.
Advancements in fusion proteins and multi-domain specific therapeutics mean progress in analytical techniques is also required. The traditional model for ADA analysis is comprised of a tiered assay approach including qualitative screening and confirmation tiers alongside a semi-quantitative titre tier. Additional characterisation of ADA positive samples is expected by regulators. Typically this is assessing them in a neutralising antibody assay to determine if they will affect the efficacy of the therapeutic, but this can now include additional tiers of analysis assessing immunogenicity against specific therapeutic domains which requires additional assay development. Likewise, the long half-life and high dosing regimens of large molecule therapeutics can make detecting ADAs problematic. High levels of drug within a sample often mean additional purification procedures are required to enable the detection of low levels of ADA. Our scientists at ACM Global Laboratories are experts in advanced ADA assay techniques such as ACE, SPEAD and PanDa which, alongside the gold-standard MSD bridging format, means we can meet the sensitivity and drug tolerance regulatory requirements for a wide range of novel therapeutics.
The advent of antibody drug conjugates (ADC) has been another huge development in large molecule bioanalysis. ADC is commonly comprised of a monoclonal antibody and cytotoxic ‘payload’ joined by a chemical linker. These harness the specificity of monoclonal antibodies to deliver a cell-killing agent directly to a cancerous target without affecting healthy tissues. The first ADC treatment was approved by the FDA in 2001, and there are currently 14 licensed in the U.S. with an estimated global market cap of nearly $8 billion; this is estimated to grow over 10% annually until 2030. Many of the original cancer-targeting monoclonal antibodies such as Trastuzumab are being reengineered into ADCs. Performing bioanalysis for ADCs requires a combination of techniques. Typically, quantification of the whole drug/antibody and ADA analysis are performed by ligand binding assays and quantification of the payload/linkers is performed by liquid chromatography mass spectrometry (LC-MS/MS). Offering both LC-MS/MS and ligand binding assay service lines means ACM Gloab Laboratories can provide the full analytical package of bioanalysis to support ADC submissions.
LC-MS/MS techniques and technology has also been developing over the last decade which has made the platform a viable alternative to ligand binding assays for protein quantification in some situations. However, the negative cost and time implications along with the prohibitive equipment requirements mean ligand binding assays remain at the forefront of large molecule bioanalysis.
Large molecule therapies made up 46% of FDA approvals in 2022 and current market trends indicate this will only increase over the next decade. This is supported by a plethora of exciting new innovative therapeutic molecules currently under development covering a wide range of modalities. The most efficient and cost-effective solution for the bioanalysis of these molecules continues to be ligand binding assays, demonstrating they’re here to stay for long into the future.