Chromatography in Biologics Manufacturing: Science, Purpose, and Resin Applications
Chromatography is a critical purification technique used in many evolving fields of biologics manufacturing including monoclonal antibodies, vaccines, and recombinant protein products. The success of a biologic drug hinges not only on its therapeutic efficacy but also on the precision and reliability of its manufacturing process—an area where chromatography plays a central role.
Scientific Basis of Chromatography in Biologics
At its core, chromatography is a separation technique based on differential interactions between components in a mixture and two phases: a stationary phase (often a resin-packed column) and a mobile phase (a liquid buffer that flows through the column). In biologics, the technique exploits subtle differences in physicochemical properties—like charge, size, hydrophobicity, or binding affinity—of the biomolecules.
When a mixture of biomolecules (e.g., harvested cell culture fluid) is passed through a chromatography column, each molecule interacts with the stationary phase differently:
- Molecules with stronger interactions elute later.
- Molecules with weaker interactions elute earlier.
This allows for selective isolation of the target biologic (the active pharmaceutical ingredient, or API) from impurities such as host cell proteins (HCPs), DNA, aggregates, or endotoxins.
Chromatography is a high-resolution and scalable technique, making it indispensable in both research and commercial-scale biologics production.
Role of Chromatography in Biologics Manufacturing
Chromatography is used extensively throughout the downstream processing (DSP) stage of biologics manufacturing. After upstream cell culture and harvest, the product-containing fluid undergoes a series of purification steps to meet regulatory requirements for purity and safety.
Key Purposes of Chromatography in DSP:
- Capture and Initial Purification:
- The first chromatographic step, often referred to as the “capture step,” isolates the biologic product from the complex culture medium.
- This step removes a large portion of host cell impurities.
- Intermediate Purification:
- Further removes specific contaminants such as process-related impurities and product variants (e.g., truncated forms).
- Polishing:
- Final chromatographic steps that refine purity and homogeneity.
- Often used to remove aggregates, viral contaminants, or charge variants.
- Viral Clearance and Impurity Reduction:
- Chromatography is often used in combination with other techniques (e.g., viral filtration), but plays a central role in achieving the purity profile demanded by regulatory agencies.
Chromatography Resin Types and Their Functions
Chromatography resins are solid-phase materials designed to interact selectively with biologic molecules. These resins vary in chemical functionality, matrix composition, and pore structure, and are tailored to achieve specific types of separation.
Resin selection is influenced by:
- Target molecule characteristics (size, charge, hydrophobicity)
- Impurity profile
- Scale and throughput requirements
- Regulatory and leachables/extractables profiles
Below are the primary categories of chromatography resins used in biologics manufacturing:
1. Protein A Affinity Resins
- Primary Use: Capture of monoclonal antibodies (mAbs).
- Mechanism: Protein A ligands on the resin specifically bind to the Fc region of IgG antibodies.
- Use Case: First step in mAb purification, commonly used in therapeutic antibody production.
2. Ion Exchange Chromatography (IEX)
a. Cation Exchange (CEX) Resins
- Mechanism: Negatively charged resins attract and bind positively charged (basic) proteins.
- Use Case: Often used after Protein A capture to remove variants or HCPs.
b. Anion Exchange (AEX) Resins
- Mechanism: Positively charged resins attract negatively charged (acidic) impurities.
- Use Case: Polishing step to remove DNA, endotoxins, and other acidic contaminants.
3. Hydrophobic Interaction Chromatography (HIC)
- Mechanism: Exploits differences in hydrophobicity.
- Use Case: Intermediate or polishing step. Separating closely related product variants or aggregates.
4. Mixed-Mode Chromatography Resins
- Mechanism: Combine multiple interaction types (e.g., ionic and hydrophobic).
- Use Case: Problem-solving tool when traditional methods fall short. Effective at removing trace contaminants or separating charge variants.
5. Size Exclusion Chromatography (SEC)
- Mechanism: Separates molecules based on size. Larger molecules elute earlier.
- Use Case: Typically a final polishing step to remove aggregates or fragment impurities.
Choosing the Right Chromatography Strategy
Chromatography steps are carefully designed during process development, often through Design of Experiments (DoE) approaches, to maximize yield and purity while minimizing costs and process time. Regulatory considerations also influence choices, especially around viral clearance and impurity profiles.
User Requirements and Qualification Challenges in Chromatography Systems
Overview
Chromatography systems, particularly automated skids used in cGMP manufacturing, are subject to rigorous qualification and validation requirements. Organizations like ISPE (International Society for Pharmaceutical Engineering) and PDA (Parenteral Drug Association) provide detailed guidance on designing and qualifying systems in accordance with quality-by-design (QbD) and GAMP 5 principles.
User Requirements Specification (URS)
A URS outlines what the end user needs a system to do, forming the foundation for design, procurement, and qualification activities. For chromatography systems, a comprehensive URS typically addresses:
- Process Capabilities
- Flow rate range (e.g., 1–20 L/min)
- Pressure range and limits
- Gradient formation (number of inlets, accuracy, CV%)
- Column compatibility (dimensions, packing support)
- Automation & Control
- PLC and HMI requirements
- Batch control and recipe management
- Real-time monitoring and alarms
- Integration with SCADA, MES, or historian systems
- Data Integrity
- 21 CFR Part 11 compliance
- Audit trails
- Electronic signatures and access control
- Cleaning and Sanitization
- CIP/SIP capabilities
- Material compatibility with cleaning agents (e.g., NaOH, ethanol)
- Compliance and Documentation
- FAT/SAT plans
- Support for IQ, OQ, and PQ
- cGMP documentation (user manual, wiring diagrams, software documentation)
- Scalability and Flexibility
- Ability to run different recipes for multiple products
- Single-use vs stainless steel components
- Modular design for process reconfiguration
Qualification Challenges
Despite clear guidance, chromatography systems present several qualification challenges, especially when balancing flexibility with regulatory compliance.
1. Complexity of Automated Systems
Chromatography skids often include multiple pumps, valves, detectors, and sensors, with complex control logic. Ensuring full GAMP 5 Category 4 or 5 software validation can be time-consuming, particularly when custom recipes or batch control features are involved.
2. Data Integrity & 21 CFR Part 11 Compliance
Qualification must ensure that electronic records are secure, attributable, legible, and auditable. Systems must include:
- Time-stamped audit trails
- Role-based access
- Secure data storage and backup
Auditing these features requires close collaboration between engineering, IT, and QA.
3. Calibration and Sensor Qualification
In-line sensors (pH, conductivity, UV) are critical for process control and release. URS must specify acceptable calibration frequency, traceability, and real-time validation mechanisms.
4. Vendor Support and Documentation
Ensuring vendors provide complete documentation for design qualification, FAT, and SAT can be difficult. ISPE recommends robust vendor audits and alignment on documentation format and content early in the project.
5. Change Management
Chromatography systems evolve over time—new software patches, component replacements, or recipe changes. A change control process tied to the validated state is critical. PDA guidelines recommend periodic requalification or impact assessments for any modifications.
6. Single-Use vs Stainless Steel
With the growing adoption of single-use technologies, hybrid systems must accommodate different materials of construction and fluid path designs. Qualification must address extractables and leachables, integrity testing, and sterility assurance for disposables.
CAI Best Practices and Industry Guidance
Risk-Based Qualification: Focus on testing high-risk functions (e.g., data integrity, process-critical parameters). This reduces the overall amount of test document execution and minimizes the opportunity for execution error and GDP issues.
Design Review Milestones: Conduct design qualification (DQ) upfront and perform thorough reviews to ensure traceability from URS to functional specs.
Traceability Matrix: Maintain a requirement trace matrix (RTM) from URS → DQ → IQ/OQ → PQ.
Benefits also come from the use of an electronic document system that can manage traceability links across lifecycle documents, making RTM and summary report approval more streamlined.
Verify where appropriate: Avoid carrying business and other needs that may be design document verifiable requirements into qualification documents. that are linked to summary reports and system release. This minimizes the amount of in-field execution as well as the amount of document execution which reduces the likelihood of execution issues and GDP errors. All of this contributes to a reduction in review and approval workflows and increased speed to readiness.
Lifecycle Approach: Follow ISPE’s Commissioning & Qualification Baseline Guide for an integrated approach from design through operation
Conclusion
Chromatography plays a pivotal role in biologics manufacturing, enabling the purification and quality control of complex biomolecules to meet stringent regulatory standards.
As biomanufacturing scales up and diversifies, the demands on chromatography systems continue to grow—not only in terms of process performance but also compliance, data integrity, and system qualification. Following guidance from ISPE and PDA ensures that systems are fit-for-purpose, properly documented, and reliably qualified across their lifecycle. The experience that CAI has in this area can reduce risk to operational readiness timelines by guiding lifecycle document development, taking a risk-based approach to qualification, and implementing our best practices into your project efforts.