Utilizing A-TEEM™ Technology for In-Process Monitoring of Monoclonal Antibody Production

Introduction

Monoclonal antibodies (mAbs) are lab-produced molecules designed to mimic the immune system's ability to recognize and bind to specific targets, such as viruses, bacteria, or cancer cells. As proteins, mAbs are composed of amino acids. Certain amino acids, specifically tyrosine, tryptophan, and phenylalanine, are intrinsically fluorescent. This property allows fluorescence spectroscopy to be used to monitor structural and environmental changes in monoclonal antibodies, making it a valuable tool for their characterization and quality assessment during manufacturing.

Fluorescence spectroscopy is a widely used technique that enables non-destructive, real-time analysis of biological samples with high sensitivity. It is ideal for studying cellular processes, protein interactions, and molecular structures, and can be used in complex mixtures without extensive sample preparation. For over 30 years, excitation-emission matrix (EEM) fluorescence spectroscopy has been utilized to characterize or "fingerprint" biological samples. Advancements in Charge-Coupled Device (CCD) technology and improved correction methods for issues like the Inner Filter Effect (IFE) have renewed interest in EEM fluorescence spectroscopy. The HORIBA Veloci™ BioPharma Analyzer is a 2-in-1 absorbance and fluorescence spectroscopy system that leverages advanced A-TEEM (Absorbance-Transmittance fluorescence Excitation and Emission Matrix) acquisition technology to capture spectral data rapidly, with acquisition times ranging from a few seconds to a few minutes, depending on the sample.

In the field of biologics manufacturing, there is increasing interest in adopting novel process analytical technologies (PAT) to support automation. Several studies have proposed EEM fluorescence spectroscopy as a promising PAT tool. This paper presents evaluation work from CPI demonstrating A-TEEM as a suitable technique to analyze six in-process manufacturing samples of monoclonal antibodies. This work is an initial step toward implementing this technique as a PAT solution in biologics manufacturing.

Materials and Methods

The manufacturing process of monoclonal antibodies (mAbs) involves several well-defined stages to ensure product purity, safety, and efficacy. To monitor the process, samples were collected at six key points for analysis with A-TEEM.

Six Key Points for Analysis

• Cell Culture Media
• Bioreactor - Protein A load
• Protein A - Flow-through
• Protein A - Wash I
• Protein A - Wash II
• Post-viral inactivation

Figure 1: Overview of the mAb Biomanufacturing Process

The biomanufacturing process for monoclonal antibodies (mAbs) includes upstream and downstream steps leading to the final product. The process begins in the Bioreactor, where mAbs are produced (e.g., from CHO mammalian cells). This is followed by Separation, typically involving filtration. The subsequent Purification stages include Protein A chromatography, low pH viral inactivation, CIEX chromatography, and AEIX flow-through chromatography. Finally, Final Formulation involves nano-filtration and UF/DF (Ultrafiltration/Diafiltration) steps to achieve the release specification. Samples are analyzed at different purity levels: Level 3 material (lowest purity), Level 2 material (intermediate purity), and Level 1 material (highest purity).

Data and Results

Each sample was diluted to achieve an absorbance value of approximately 0.5 au. Excitation-Emission Matrix (EEM) data were collected with excitation wavelengths ranging from 239 nm to 800 nm in 3 nm increments, and emission wavelengths from 250 nm to 800 nm in 5 nm increments. The integration time was set to 0.01 seconds. Data were corrected for the inner filter effect (IFE), as well as for first- and second-order Raman scattering at 16 nm and 32 nm bandpass, respectively, and normalized to a maximum intensity of 1. Parallel Factor Analysis (PARAFAC) was conducted using Solo software (Eigenvector Research Inc., USA).

Figure 2: A-TEEM Contour Plots

This figure displays A-TEEM contour plots from the six in-process samples analyzed. The samples represent key stages in monoclonal antibody production: Cell Culture Media, Bioreactor, Protein A - Flow-through, Protein A - Wash I, Protein A - Wash II, and Post Viral Inactivation.

PARAFAC analysis returned a 4-component model that explained the raw data variance with over 98% accuracy. Each PARAFAC component corresponded to a specific biological fluorophore: tyrosine (Ex. 275 nm, Em. 300 nm), tryptophan 1 (Ex. 278 nm, Em. 330 nm), tryptophan 2 (Ex. 278 nm, Em. 350 nm), and a group comprising vitamins and co-factors (Ex. 356 nm, Em. 455 nm).

Figure 3: PARAFAC Model Component Loadings

Figure 3 presents the modelled emission, excitation, and component loadings for each of the six in-process samples. The left panel shows excitation spectra, the middle panel shows emission spectra, and the right panel shows component loadings. Notably, the data reveals a red shift in tryptophan emission in the post-viral inactivation (post-VI) purified monoclonal antibody (mAb) sample. This red shift suggests a change in the local environment surrounding the tryptophan residues, which is often used as an indicator of protein folding state.

Conclusion

In this study, A-TEEM spectroscopy was employed to characterize six in-process monoclonal antibody samples. As an analytical technique, A-TEEM offers several advantages: it is rapid, easy to implement, highly sensitive, and generates data-rich outputs. These qualities make it well-suited for use either as a standalone tool or in combination with other spectroscopic methods such as Raman, UV-Vis, or infrared spectroscopy. The successful application of A-TEEM technology in the context of monoclonal antibody production highlights its potential as a powerful Process Analytical Technology (PAT) for biologics manufacturing. This work represents an initial step toward evaluating the feasibility of A-TEEM.

Acknowledgements

HORIBA thanks CPI, in particular Dr. Daniel Myatt and Dr. Vicki Linthwaite, for their valuable work in assessing A-TEEM as an analytical tool in bioprocess measurement. This work was conducted in the context of the project "Integrating Continuous Technologies for the Rapid Delivery of Cost-Effective Biotherapeutics to Patients," funded by Innovate UK (project 93825).

References

• Myatt D. et al., "Controlling antibody in-process purification through innovative A-TEEM fluorescence fingerprinting analytics." Poster presented at AIS at AIS, Tours, 22nd June 2023.
• Myatt D. et al., "The Development of Continuous Biologics at CPI." Talk presented at BPS Europe, Barcelona, 21st March, 2025.

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