Chromatography Resin Lifetime Validation: Methods, Influencing Factors, and Case Studies

Over the past 20 to 30 years, biotechnology in China has made significant advancements, and biopharmaceuticals have gained increasing attention. The essential raw materials for biopharmaceutical production form the backbone of the entire industry, with their quality, stability, and supply capacity directly impacting drug research, production efficiency, and costs. Among these raw materials, chromatography resins play a crucial role in ensuring the purity, efficacy, and safety of biopharmaceuticals. Strict control over chromatography resins is essential to maintain the consistency of process performance and product quality.

During bioprocess purification, chromatography resins are used to isolate and purify target products. However, impurities gradually accumulate within the resin over time. The lifetime of chromatography resins is typically defined as the number of cycles a resin can undergo under specific process conditions before its performance deviates from predefined process parameters. It is assessed using key indicators such as dynamic binding capacity, host cell protein (HCP) content, nucleic acids, lipids, metabolic byproducts from the culture process, and endotoxin levels.

Necessity of Lifetime Validation

1. Cost Control: The cost of a single batch of chromatography resin can reach hundreds of thousands of RMB, extending resin lifetime can significantly reduce costs.

2. Process Robustness: Lifetime validation ensures that the performance degradation of the resin remains within a controlled range, maintaining process consistency and product quality.

3. Regulatory Compliance: Complies with GMP and FDA/EMA validation requirements for critical consumables.

Factors Affecting Chromatography Resin Lifetime

1. Type of Drug Substances

2. Type of Chromatography Resin

3. Maintenance of the Chromatography Column

4. Composition of the Chromatography System

5. Packing Quality of the Chromatography Column

6. Quality of Reagents

Establishing and Validating a Scaled-Down Model in Resin Lifetime Studies

First, a scaled-down model of the chromatography step is established, followed by corresponding experimental validation of the model. The development of this scaled-down model is based on the scaling principles of chromatography, ensuring that key parameters such as column height, linear flow rate, sample loading capacity, cleaning and elution volumes, and column efficiency remain consistent. The model confirmation requires three repeated runs, and performance parameters from the laboratory, pilot, and production scales are compared. Data shows that there are no significant differences in parameters across different scales. This scaled-down model is considered a reliable representation of production-scale performance and is suitable for process characterization studies.

The scaled-down model and the lifetime and cleaning validation of the chromatography resin can be conducted simultaneously. The validation process involves assessing factors such as impurity residue, yield, and endotoxin levels to determine whether the resin meets the required standards and maintains effectiveness as indicated by the scaled-down model. Establishing and confirming the scaled-down model is crucial for guiding large-scale chromatography operations.

Routine Cleaning and Maintenance of Chromatography Resin

1. Regeneration:

Regeneration is performed after each chromatography cycle to restore the original functionality of the resin. The choice of buffer depends on the resin type. For example, ion exchange resin typically use 2 M NaCl, while hydrophobic interaction resin generally use purified water or a low-concentration buffer.

2. CIP (Cleaning-in-Place):

CIP removes impurities that are not eliminated during regeneration and helps prevent the accumulation of contaminants. It is typically carried out every 1 to 10 cycles. Appropriate cleaning solutions should be selected according to the product manual for each specific type of chromatography resin.

3. Sanitization:

Sanitization is carried out between production batches to eliminate microbial contamination in the chromatography resin. A common method involves using 0.5–1 M NaOH for 30–60 minutes. For resin that are not compatible with NaOH, alternative sanitization methods should be used. The purpose is to prevent microbial growth and remove endotoxins.

4. Storage:

Storage aims to prevent microbial growth and endotoxin contamination of the chromatography resin during non-continuous operations. Storage conditions should be chosen based on the characteristics of the resin, typically using 20% ethanol or 0.01 M NaOH. Proper storage is crucial for maintaining the resin’s lifespan.

Primary Contaminants Removed During Chromatography Resin Cleaning

Chromatography resin cleaning targets various types of contaminants, each requiring specific cleaning strategies. Common contaminants include soluble host cell proteins, denatured proteins, hydrophobic proteins, lipids, nucleic acids, pigments, endotoxins, and microorganisms.

Chromatography Resin Cleaning Validation

During the cleaning validation process, a blank run is performed to verify the effectiveness of the cleaning procedure. In this process, the equilibration buffer is loaded onto the column in place of the actual sample, and the resulting eluate is collected. The collected fractions are analyzed for residual impurities and their concentrations to assess whether the cleaning procedure meets the required standards. The blank run helps confirm the effectiveness of cleaning and prevents the risk of impurity accumulation over time.

Case Study 1: Lifetime Validation of MaXtar® Protein A Affinity Chromatography Resin by BioLink

Drug Substances: Clarified cell culture supernatant containing monoclonal antibodies, post-depth filtration.

Study Design: A total of 200 purification cycles were performed using MaXtar® Protein A affinity resin, following a standard monoclonal antibody (mAb) platform process. Sanitization was carried out using 0.1 M NaOH for 30 minutes per cycle, with the concentration increased to 0.5 M NaOH after 140 cycles. After each cycle, resin regeneration was performed. Every 10 cycles, key performance indicators were assessed, including dynamic binding capacity (DBC), yield, SEC profile, HCP, and rProtein A shedding.

Results:

• Yield remained above 80% after 200 cycles, meeting expected performance standards.

• Dynamic Binding Capacity (DBC) retained over 80% of its initial value.

• SEC analysis showed no significant changes in product purity.

• HCP residuals were maintained below 1000 ppm.

• rProtein A Shedding was kept below 25 ppm.

Case Study 2: Lifetime Validation of BioLink Anion Exchange Resin for Viral Clearance

In anion exchange chromatography, the resin’s ability to remove Murine Leukemia Virus (MuLV) and Minute Virus of Mice (MVM) was evaluated across multiple reuse cycles. The performance was assessed at early, mid, and late stages of the resin’s lifetime. Prior to loading, the feed samples were properly conditioned to meet the requirements for effective viral binding and clearance.

Experimental results demonstrated that the viral clearance capability was not affected by up to 150 reuse cycles of the resin.

Introduction to BioLink Suzhou Application Center

The BioLink Suzhou Application Center covers an area of 3,000 square meters and is equipped with an R&D office area, cell culture laboratory, microbiology laboratory, purification laboratory, pilot-scale workshop, and analytical laboratory. The center is dedicated to providing customers with upstream and downstream process development, small- and pilot-scale sample preparation, resin lifetime validation, and technical training services.