Enhancing Biopharma QC: The Role of Advanced Detectors
Why is there rapid growth in protein therapies like mAbs?
Since the first FDA-approved recombinant protein-based therapy in 1982, biologics have continued to hold immense promise due to their specificity and ability to target various mechanisms of action (MoA) (1). These MoAs include, but are not limited to, protein degradation, hormone replacement, immunological cell signaling, and neutralization of cytokines. Because of their versatility there are more than 890 FDA-approved therapeutic proteins of all classes including approximately 350 mAb therapies (2). Despite their success, these therapies face challenges due to their susceptibility to aggregation, degradation, and denaturation, which are intrinsically linked to their therapeutic activity through the structure-function relationship. These properties must be controlled during the manufacturing process and monitored in quality control (QC) labs.
Why are aggregates a risk?
Aggregation and size variant analysis is a particular critical risk factor (3,4). As previously mentioned, the structure of biotherapeutics is intrinsically related to their function. Not only that aggregation decreases the overall available therapeutic molecules and that effect dosage, but aggregates can also trigger innate immune response by non-specific binding. This could lead the body to recognize these beneficial therapeutics as dangerous, leading to the creation of anti-drug antibody (ADA). One would find that many regulatory bodies create guidelines to quantify the risk of aggregation. For example, USP general chapter <129>,<621>,<697>,<787>,<788>,<855>,<1049> in some for or another involve addressing the risk and quantify aggregate formation.
Much effort in the development process is spent on spent on controlling aggregation, self-assembly, and oligomerization is achieved by optimizing the formulation. During the formulation development stage excipients such as polysorbates are added for suspension stability while sugars are added for cryoprotectants (5). Additionally, adjusting the ionic strength and pH through the addition of amino acids, acids, or bases to the buffer system helps to prevent or support self-assembly for each unique therapy. (5). Even with this attention given to formulation during development, stability issues can still arise during manufacturing and storage which are closely monitored during QC.
Why are some analytical instruments selected for QC?
Liquid Chromatography (LC) wasn’t always a standard instrument in the protein QC lab. Its adoption was significantly driven by the need for more precise and reliable testing methods and can be linked to the first recombinant product produced, insulin (6) In the early days of insulin production, testing methods were dominated by immunoassays that ultimately didn’t match the modernization this drug went through itself as it progressed from extraction to manufacture in a bioreactor using recombinant technology. The introduction of LC revolutionized not only insulin testing but the broader biopharma testing by providing a more accurate and efficient method for analyzing insulin identity, purity, potency and degradation products (7,8,9) This shift was crucial in ensuring the safety and efficacy of insulin, leading to its widespread adoption in laboratories around the world due to HPLCs.
Ensuring robust methods for Quality Control (QC) involves optimizing and assessing method parameters such as sample preparation, injection, conditions, and data analysis. Key considerations include the system suitability criteria, consistent supply of critical reagents, and thorough training of QC analysts. Effective communication between development and QC labs, along with strategies to manage vendor-driven changes, are crucial for successful method transfer.
What are the needs in QC today?
Regulatory agencies have established strict guidelines for monitoring and controlling aggregates and particles. Protein aggregates, regardless of their size, impact the safety and efficacy of biologic therapies. Forced chemical degradation either by hydrolysis, oxidation, photolysis or thermal stress is essential for ensuring safety and efficacy throughout the shelf-life of a therapy. A simple table summarizing the more common conditions and durations for forced degradation tests are listed in the table below. (10, Table 1)
Here's a table summarizing an example of conditions and durations for the forced degradation tests:
| Degradation Type | Conditions | Duration |
|---|---|---|
| Oxidation | - Hydrogen peroxide (0.1% to 3%) - Other oxidizing agents (e.g., metal ions) |
Up to 7 days or until 20% degradation |
| Photolysis | - UV and visible light - Minimum 1.2 million lux hours - 200 watt-hours/m² of UV light |
Several days |
| Thermal Stress | - Dry heat and wet heat - Temperatures: 40°C to 80°C |
Multiple time points (e.g., 1, 3, 5 days) |
| Hydrolysis | - Acidic: HCl or H₂SO₄ (0.1 M to 1 M) - Basic: NaOH or KOH (0.1 M to 1 M) |
Up to 7 days at elevated temperatures (40°C to 60°C) |
In addition to the standard HPLC analysis, Size exclusion chromatography and multi-angle light scattering (SEC-MALS, e.g. DAWN™ or miniDAWN™ MALS instruments) offers orthogonal confirmation by measuring absolute molecular weight, size (Rg), size distributions, fragments and non-covalent associations. A significant advantage is that it doesn’t require additional acquisition time, as it operates downstream from the UV or PDA detector, utilizing the same HPLC technique already in use.
When considering higher order species, aggregates aren’t the only concern. Oligomers, which can be functional like insulin (SEC-MALS app note) or an unwanted step towards aggregation, also matter. The higher-order of the drug substance is critical due to the structure-function relationship or structure activity relationship (SAR) of biologic therapies. SAR is a quality attribute that can be classified as a critical quality attribute (CQA), making it essential to include in extended characterization or release assays in the QC lab to better understand the biophysical properties of product.
Why should MALS be incorporated into QC processes?
Incorporating SEC-MALS into QC processes address both aggregation and SAR questions, providing extended characterization information. MALS, coupled with SEC, is an established method for effective protein quality control and aggregate detection. This technique accurately determines absolute molecular weight and size distributions, and radius of gyration (Rg), all of which are quality attributes for biotherapeutics. By detecting and quantifying aggregates and providing insights into size and structure, MALS helps ensure the safety and efficacy of innovator products and biosimilars.
Many scientists already use MALS in protein QC to qualify their standards, and companies that sell standards often market them as QC qualifiers. Testing at release, including MALS, ensures that any changes in the protein’s structure are detected before the product reaches the patient. USP <1430> “Analytical Methodologies Based on Scattering Phenomena" provides further guidance on these techniques.
Often, release assays favor simplicity and do not include advanced characterization and detection by sophisticated techniques. As a result, the most accurate and detailed information is not captured during release. However, many labs ensure thorough characterization by incorporating extended tests, such as SEC-MALS for size variant analysis. While SEC-MALS may not be part of the release assays, it can still be utilized in QC labs for extended characterization.
How can software enable instrument adoption in QC?
The adoption of new techniques is primarily driven by the need for safety, more accurate measurements, and ease of use. Additionally, providing a complete view of the sample the first time helps avoid additional work following potential deviations at release. Ease of use and QC readiness are crucial for successful adoption.
Another key factor is the desire to decrease risk and improve efficiency in demanding labs. For broader QC adoption, incorporating MALS into Empower™ Chromatography Data System (CDS) lowers the barrier by reducing a lab’s digital footprint and efforts for validation from two software system to one. Empower CDS is a mainstay in QC labs, offering efficient and secure data acquisition, management, processing, and reporting. It can scale from a single workstation to an enterprise-wide network, allowing for connectivity across sites and centralized data management, and is the industry leader in data integrity. Empower CDS ensures compliance with regulatory standards and is an enabling driver towards the successful adoption of advanced instruments in QC labs. According to Waters data estimates, 80% of novel drugs submitted to the FDA, EMA, and China NMPA use Empower CDS (11).
Conclusion
Biologic therapies have revolutionized medicine by targeting specific mechanisms of action, offering treatments for various diseases. Despite their promise, these therapies face challenges at release in part due to their strong structure-function relationship that can be disrupted due to aggregation, degradation, and partial unfolding. Advanced techniques SEC-MALS are essential for ensuring safety and efficacy by accurately measuring critical quality attributes and providing extended characterization tests that further enhance confidence in the product. As biologics evolve, adopting sophisticated QC methods and systems under Empower CDS reduces the barrier of adoption in QC and allows scientists to focus on ensuring the production of high-quality therapies that benefit patients worldwide.
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References
Biologic therapies have revolutionized medicine by targeting specific mechanisms of action, offering treatments for various diseases. Despite their promise, these therapies face challenges at release in part due to their strong structure-function relationship that can be disrupted due to aggregation, degradation, and partial unfolding. Advanced techniques SEC-MALS are essential for ensuring safety and efficacy by accurately measuring critical quality attributes and providing extended characterization tests that further enhance confidence in the product. As biologics evolve, adopting sophisticated QC methods and systems under Empower CDS reduces the barrier of adoption in QC and allows scientists to focus on ensuring the production of high-quality therapies that benefit patients worldwide.
- Nat Commun. 2023. Apr 27: 14:2411. Doi: 10.1038/s41467-023-38039-x
- Drug Discovery Today. Vol 29, No. 7, Jul 2024
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- Lundhal, M., Fogli, S. Colavita, P. & Scanlan, E. Aggregation of protein therapeutics enhances their immunogenicity: causes and mitigation strategies RSC Chem Biol. 2021 May 4;2(4):1004–1020. doi: 10.1039/d1cb00067e
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- “100 Years of Insulin” FDA https://www.fda.gov/about-fda/fda-history-exhibits/100-years-insulin
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- Hansen, M., & Pedersen, S. (2012). High-performance liquid chromatography in the analysis of insulin and its analogues. Journal of Chromatography B, 879(13-14), 1154-1161. doi: 10.1016/j.jchromb.2011.12.034.
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- Ruchi Et al. Development of forced degradation and stability indicating studies of drugs—A review, Journal of Pharmaceutical Analysis, Volume 4, Issue 3,2014, Pages 159-165,ISSN 2095-1779, https://doi.org/10.1016/j.jpha.2013.09.003. (https://www.sciencedirect.com/science/article/pii/S2095177913001007)
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