In-line Fiber Optic Sensor for Real-time Detection of IgG Aggregates in Affinity Chromatography

Abstract:

Therapeutic monoclonal antibodies (mAbs), particularly Immunoglobulin G (IgG), are extensively used in treating a variety of diseases. However, their production often faces significant challenges, primarily due to aggregation during the manufacturing process. This aggregation negatively impacts the safety and quality of the product, making the detection and removal of aggregates critical. Chromatography plays a key role in mAb purification, but conventional systems lack efficient real-time monitoring for both the product and its aggregates. This study introduces a novel in-line fiber-optic sensor based on localized surface plasmon resonance (LSPR) to detect IgG and its aggregates during affinity chromatography. The sensor, placed at the outlet of a Protein A column, detects IgG aggregates at concentrations below 1%, with a sensitivity capable of distinguishing differences as small as 0.3% between samples. The slower dissociation rates of aggregates, compared to monomers, allow for clear differentiation. This real-time, in-line detection approach significantly improves automation and process optimization in biomanufacturing, offering substantial advantages over traditional off-line analysis.

1. Introduction

Since the approval of the first therapeutic monoclonal antibody (mAb) in 1986, mAbs, particularly Immunoglobulin G (IgG), have become integral in treating various diseases. However, the production of mAbs is complex, costly, and prone to aggregation, which can severely affect both the product’s safety and therapeutic efficacy. The increasing demand for continuous biomanufacturing, supported by frameworks such as Quality by Design (QbD) and Process Analytical Technology (PAT), aims to streamline production processes. Despite these advancements, the absence of real-time, in-line monitoring tools remains a major obstacle. Traditional methods of detecting aggregates are often time-consuming, labor-intensive, and require expensive off-line equipment. Protein A-based affinity chromatography is commonly used to purify IgG, but separating aggregates from monomers can be challenging, particularly when both co-elute. To overcome this challenge, a fiber-optic sensor based on localized surface plasmon resonance (LSPR) was developed to monitor IgG monomers and aggregates in real-time. This technology enhances the ability to optimize, intensify, and automate biopharmaceutical purification processes.

2. Materials and Methods

2.1 Sensor Chip Preparation

Carboxylated sensor chips (ArgusEye AB, Sweden) were activated with a 1:1 mixture of 0.4 M EDC and 0.1 M NHS for 40 minutes, followed by rinsing with MilliQ water. Protein A (0.5 mg/mL) was then coupled to the activated chips for 2 hours, after which unreacted carboxyl groups were deactivated using 1 M ethanolamine (pH 8.5) for 30 minutes. The sensor chips were stored in PBS buffer until further use.

2.2 Chromatography Experiments

2.2.1 Affinity Chromatography with pH Gradient Elution

Chromatography experiments were carried out using an AKTA Pure system (Cytiva) and a 2 mL MabSelect PrismA column. Clarified samples containing 2.6 mg/mL IgG were loaded for 32 minutes. The column was equilibrated with 10 mM PBS (pH 7.4) before loading, followed by washing and elution with pH gradient citrate buffers (pH 4-3 and pH 4.5-3). A stripping step and a Clean-in-Place (CIP) procedure with sodium hydroxide were applied post-elution. The dynamic binding capacity of the MabSelect PrismA column was determined to be approximately 80 mg IgG/mL resin at a 6-minute residence time.

2.2.2 Affinity Chromatography with Isocratic Elution

An AKTA Pure system and a 1 mL MabSelect PrismA 25 chromatography column were used for isocratic elution experiments. The column was equilibrated and washed with PBS, followed by isocratic elution with 50 mM citrate buffer at pH 3.5 or 3.0. Elution was followed by a stripping step, and the column was re-equilibrated with PBS.

2.2.3 Size Exclusion Chromatography (SEC) Analysis

Size exclusion chromatography (SEC) was employed to confirm the presence of aggregates in elution pools. Two SEC systems were utilized: SEC-UV and SEC-MALS-RI-UV. SEC-UV was performed using a Superdex 200 Increase 10/300 GL column, while SEC-MALS-RI-UV utilized multi-angle light scattering and refractive index detectors to determine the presence and size distribution of aggregates.

3. Results and Discussion

3.1 In-line Monitoring of Protein A Chromatography with pH Gradients

In Protein A affinity chromatography, pH gradient elution improves aggregate separation. The LSPR sensor demonstrated high sensitivity, detecting IgG monomers and aggregates in real-time, even at low aggregate concentrations. During the loading phase, the LSPR sensor exhibited a rapid baseline shift due to the sample’s strong color, but no breakthrough was observed in the column. The sensor performed well throughout the washing and buffer exchange steps. The LSPR sensor showed a higher binding response with a pH gradient from 4 to 3 compared to the gradient from 4.5 to 3, effectively detecting aggregates, particularly with the faster gradient.

3.2 Comparison of UV and LSPR Signals for Monomers and Aggregates

In larger chromatography setups, the LSPR sensor outperformed the UV detector in early aggregate detection during elution. The LSPR signal showed a sharp rise during the elution of aggregates, confirmed by off-line SEC analysis, while the UV signal exhibited a weaker response, particularly at higher IgG loadings. The LSPR sensor maintained superior sensitivity and specificity.

3.3 LSPR-Based Detection of Aggregates During Isocratic Elution

Under isocratic elution conditions, the LSPR sensor was able to detect IgG aggregates that were undetectable by UV signals. The sensor differentiated between aggregates and monomers based on dissociation rates, providing a sensitive method for real-time aggregate monitoring under challenging isocratic conditions.

4. Conclusion

The in-line LSPR sensor offers a highly sensitive, real-time method for monitoring critical quality attributes (CQAs) like IgG monomers and aggregates during affinity chromatography. The sensor can detect aggregate concentrations as low as 0.3%, providing significant advantages over traditional UV detectors, especially during complex chromatography processes. The LSPR sensor’s ability to differentiate aggregates from co-eluting monomers demonstrates its potential to enhance process efficiency, automation, and product quality monitoring in biopharmaceutical manufacturing.

Acknowledgments

The authors acknowledge the funding from the European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant No. 841373) and the Swedish Innovation Agency (VINNOVA). They also thank the staff at Testa Center, Uppsala, Sweden, for their assistance with chromatography measurements, as well as Dr. Mats Nilsson and Kristoffer Rudenholm Hansson from BioInvent International AB for providing IgG samples and for their valuable discussions.