Biopharmaceutical Testing

Evers, Dirk-H., et al. Journal of Chromatography B 1174 (2021): 122717.

In biopharmaceutical formulations, the enzymatic hydrolysis of polysorbates, particularly Polysorbate 20 (PS20), is a known risk factor for particle formation, which can compromise product stability. This study presents a novel ultra-performance liquid chromatography with quadrupole detector (UPLC-QDa) method to quantify free fatty acids (FFAs) released from the hydrolysis of PS20, a key factor in particle formation.

The developed method is label-free, directly measuring ionized FFAs, and is capable of determining both the percentage of ester bond hydrolysis and the absolute concentration of released fatty acids in ng/mL. This novel technique utilizes an isolator column to enhance sensitivity by removing trace fatty acids from the eluents, making it ideal for detecting even low levels of degradation.

The method was validated through case studies involving monoclonal antibodies (mAbs) in PS20-containing formulations, revealing distinct fatty acid release patterns that correlate with hydrolysis stages. The approach provides a reliable tool for monitoring PS degradation during the development of biologic formulations, offering both external calibration and recalculation options for concentration determination.

This method significantly aids in the prediction and prevention of particle formation, making it a valuable quality control tool in biopharmaceutical manufacturing, especially for ensuring the stability and shelf life of liquid biologic formulations.

Massei, A., et al. European Journal of Pharmaceutics and Biopharmaceutics (2024): 114342.

The production of antibody-based biopharmaceuticals has grown significantly over the past few decades, but these molecules are highly sensitive to stresses during manufacturing, leading to structural changes such as aggregation, fragmentation, and oxidation. Detecting and understanding these alterations is critical for maintaining product quality and preventing out-of-spec results.

This study presents Raman spectroscopy coupled with Principal Component Analysis (PCA) as an effective, non-invasive tool to characterize monoclonal antibodies (mAb) under various stresses. The IgG1 fusion protein was subjected to thermal stress (50 °C for one month) and oxidative stress (exposure to hydrogen peroxide), and changes in the protein structure were monitored. Raman spectra revealed key bands that correlated with changes in the protein's secondary and tertiary structures, specifically in the amino acid regions and amide bands.

Thermal stress led to a decrease in amino acid signal intensity, while oxidation caused significant alterations in the C–H stretching region. Notably, PCA analysis allowed for the discrimination between different stress conditions and even between mAb and hormone-based products, showcasing the robustness of the method.

This Raman spectroscopy-based approach is not only minimally invasive and rapid but also offers a reliable method for detecting structural changes in protein-based biopharmaceuticals, ensuring product stability and quality throughout manufacturing.

Joshi, Srishti, Kratika Upadhyay, and Anurag S. Rathore. Journal of Chromatography B 1212 (2022): 123511.

Charge heterogeneity in monoclonal antibodies (mAbs) is a key parameter in biopharmaceutical analysis, influencing product stability and efficacy. Traditional charge variant separation is commonly achieved using cation-exchange chromatography (CEX) with UV detection at 280 nm. However, the sensitivity of UV-based detection can be limiting, especially for low-abundance charge variants.

This study evaluates the potential of fluorescence detection (FLD) as a more sensitive alternative to UV detection in CEX for mAb charge variant analysis. By utilizing a mass-spectrometry-compatible, pH-based CEX method with volatile ammonium acetate salt, the study demonstrated that FLD achieved significantly lower limits of detection (LOD) and quantification (LOQ) compared to UV detection—59 times lower for both parameters. The linearity of FLD detection ranged from 1 to 200 µg, with excellent accuracy and reproducibility.

The FLD-CEX method was successfully applied to directly profile charge variants in CHO cell harvest supernatant and assess the biosimilarity of trastuzumab biosimilars, showing its robustness for Process Analytical Technology (PAT) applications. This sensitive, validated method provides an efficient and reliable tool for detailed biopharmaceutical analysis, enabling precise charge variant characterization and facilitating biosimilarity assessment in mAb product development.