Advancing Biotherapeutic Analysis with Avantor® ACE® Exclude SEC Columns

Overview

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• Source: VWR


• Date: 05 Oct,2026

The development of biotherapeutics requires the purification of proteins from numerous impurities. Size Exclusion Chromatography (SEC) is a powerful method of purification and analysis of biotherapeutics. During analysis of biotherapeutics, the method of choice is often SEC. The ACE Exclude SEC column is made for separation of biomolecules and offers reproducible and repeatable separation.

Challenges in Conventional SEC Analysis:

Despite its importance, conventional SEC methods often face limitations such as:

1. Inconsistent separation between monomer and aggregates
2. Variability between column batches affecting method robustness
3. Limited column lifetime under high-throughput conditions
4. Secondary interactions requiring high salt concentrations

This causes increased costs, late answers to validation questions, and hesitation to trust analytical results.

Solution Overview:

Avantor^® ACE^® Exclude SEC columns were developed with features intended to address these limitations through optimized particle and pore technology.  With a selectivity of 100-800 kDa, the column has been shown to be lot-to-lot reproducible with different manufacturing lots. The column is used to separate mAbs, antibody-drug conjugates (ADCs) and other therapeutic proteins.

Key Benefits and Value Proposition:

Based on internal testing, the Avantor^® ACE^® Exclude SEC demonstrated:

1. Fewer column replacements
2. Reduced downtime
3. Lower cost per analysis
4. Improved laboratory productivity

Technical Performance:

Robust Across Conditions

The column demonstrated consistent chromatographic performance

under both low and high salt mobile phase conditions, providing flexibility in method development.

Optimized Design Specifications

1. Particle size: 3 µm
2. Pore size: 250 Å
3. Selectivity: 100–800 kDa
4. pH stability: 2–8
5. Compatible with standard phosphate buffer systems

Application Example: mAb Aggregate Analysis:

SEC plays a critical role in:

1. Protein purity assessment
2. Aggregate detection
3. Structural integrity monitoring
4. Supporting regulatory compliance

In a demonstrated application, the ACE^® Exclude SEC column successfully separated NISTmAb monomer from induced aggregates, confirming its suitability for demanding biopharmaceutical analyses. Figures 1 shows that the Avantor ACE Exclude SEC columns provide consistent separation of protein standards across different lots.

Figure 1

Test Conditions Mobile phase: 20 mM Na₂HPO₄ (pH 7.0) with NaCl Flow rate: 0.35 mL/min Temperature: 25 °C Injection volume: 0.5 µL Sample: Thyroglobulin γ-Globulin Ovalbumin Myoglobin Vitamin B₁₂

mAb Aggregate Separation Application

In biopharmaceutical development, SEC plays a critical role in:

1. Assessing protein purity
2. Detecting aggregates
3. Monitoring structural integrity
4. Supporting quality control and regulatory compliance

SEC is widely applied in the characterization of monoclonal antibodies (abs), antibody–drug conjugates (ADCs), and other therapeutic proteins, supporting quality control and regulatory compliance throughout the drug development lifecycle.

In this example application, NISTmAb at 1 mg/mL was diluted 1:9 in mobile phase and heated to 65 °C for 30 minutes with agitation to induce aggregate formation. As shown in Figure 2, the Avantor ACE Exclude SEC column successfully separated the NISTmAb monomer from the aggregates.

Figure 2

Test Conditions Column: Avantor ACE Exclude SEC Particle size: 3 µm Dimensions: 300 × 4.6 mm Mobile phase: 100 mM Na₂HPO₄ (pH 7.0) with 200 mM NaCl Flow rate: 0.35 mL/min Temperature: 25 °C Injection volume: 1 µL Detection: UV (220 nm) Sample: 1 mg/mL NISTmAb, diluted 1:9 in mobile phase

Conclusion

The Avantor^® ACE^® Exclude SEC column provides a reliable SEC platform to analyze biotherapeutics. SEC methods often suffer from poor reproducibility and subpar column lifetime.

The ACE^® Exclude SEC column provides resolution and reproducibility to enhance your SEC methodology. The column is designed to accelerate analysis of monoclonal antibody (mAb) aggregates, providing an opportunity to increase analysis throughput while reducing analysis cost. The ACE® Exclude SEC column can also be utilized to analyze other biotherapeutics in addition to mAbs.