PERLs from Purification Resins in Biopharma

Understanding the PERLs Threat to Biologics

Process equipment related leachables can impact biopharmaceutical products in ways that small molecule drugs typically don’t experience:

Binding to active sites: Leached compounds might bind to the therapeutic protein’s active site, the specific region responsible for the drug’s mechanism of action. This blocks the intended biological activity, reducing drug efficacy. Even if only a small percentage of protein molecules are affected, this can significantly reduce the effective dose delivered to patients.

Protein denaturation: Some leached compounds can disrupt the non-covalent interactions (hydrogen bonds, hydrophobic interactions, electrostatic forces) maintaining protein three-dimensional structure. When proteins denature, they lose their functional conformation and biological activity. This can occur during processing, storage, or after administration, potentially rendering the drug product ineffective.

Aggregation promotion: Certain leached compounds can promote protein aggregation, where multiple protein molecules clump together. Aggregates are not only less effective therapeutically but can trigger immune responses in patients, creating safety concerns beyond simple loss of efficacy.

Direct patient risk: Some PERLs may themselves be toxic, allergenic, or otherwise harmful to patients. This represents a traditional safety concern similar to leachables from container closure systems, but originating from manufacturing equipment.

These risks are particularly concerning for biopharmaceuticals because:

• Protein drugs are typically administered parenterally (injected), providing direct systemic exposure
• Biologics often treat serious or chronic conditions requiring repeated dosing
• The mechanisms of protein-PERLs interactions can be complex and difficult to predict
• Effects may be concentration-dependent and not immediately obvious during development

The Growing Importance as Biopharma Expands

The biopharmaceutical industry continues rapid growth. Monoclonal antibodies, therapeutic proteins, peptides, vaccines, and cell and gene therapies represent increasing portions of pharmaceutical pipelines and approved products.

This expansion means:

• More manufacturing facilities using purification resins and other process equipment
• Greater diversity of biologic drug modalities with varying sensitivities to PERLs
• Increased patient populations receiving biologic therapies
• Heightened regulatory scrutiny of manufacturing process controls

As the industry scales, systematic approaches to understanding and controlling PERLs become not just good practice but essential for maintaining product quality across expanding manufacturing operations.

Purification Resins: Essential but Potential Sources

Chromatography purification is fundamental to biopharmaceutical manufacturing. After initial expression in cell culture (bacterial, yeast, or mammalian cells), the target protein exists in complex mixtures with host cell proteins, DNA, endotoxins, and culture media components.

Purification typically involves multiple chromatography steps using different resin types:

• Affinity chromatography: Highly selective binding based on specific biological interactions (e.g., Protein A for antibodies)
• Ion exchange chromatography: Separation based on charge differences between target protein and contaminants
• Hydrophobic interaction chromatography: Separation based on hydrophobicity differences
• Size exclusion chromatography: Separation based on molecular size

Each resin type consists of a base matrix (often cross-linked agarose or synthetic polymers) with functional groups providing the separation mechanism. These resins contact process streams repeatedly across many manufacturing batches.

The challenge: resins are complex materials containing:

• Base matrix components and cross-linkers
• Functional ligands (affinity tags, ion exchange groups, hydrophobic moieties)
• Stabilizers and preservatives
• Residual synthesis reagents
• Potential degradation products

Any of these might leach into process streams, especially under the cleaning, sanitization, or extreme pH conditions used during operation and resin regeneration.

The Multi-Technique Analytical Strategy

Joel’s approach recognizes that PERLs span enormous chemical diversity, requiring multiple complementary techniques for comprehensive profiling:

High-Resolution LC-MS (Liquid Chromatography-Mass Spectrometry):

• Detects semi-polar to polar organic compounds
• High-resolution accurate mass enables molecular formula determination
• Provides structural information through fragmentation
• Covers broad molecular weight range

GC-MS (Gas Chromatography-Mass Spectrometry):

• Detects volatile and semi-volatile organic compounds
• Complementary to LC-MS for less polar, thermally stable species
• Excellent for identifying unknowns through spectral library matching
• Sensitive detection of low-level volatile contaminants

ICP-MS (Inductively Coupled Plasma-Mass Spectrometry):

• Detects inorganic elements and elemental impurities
• Parts-per-billion sensitivity for trace metal analysis
• Covers broad elemental range from lithium to uranium
• Critical for identifying metal catalysts, stabilizers, or degradation products

This multi-technique approach ensures that organic compounds (polar and non-polar, volatile and non-volatile) plus inorganic species are all captured in the extractables profile.

Worst-Case Extraction Conditions

Extractables studies intentionally use exaggerated conditions compared to normal manufacturing to reveal the full range of compounds that could potentially leach. Joel’s study employed worst-case conditions including:

• Extended contact times exceeding typical process durations
• Elevated temperatures promoting extraction
• Aggressive solvents representing extreme manufacturing conditions
• Multiple extraction cycles simulating repeated resin use

These conditions intentionally over-extract compared to normal operations. The philosophy: better to identify all potential extractables during development, even if they wouldn’t normally leach at significant levels, than to miss compounds that might appear under occasional process variations or after extended resin use.

The Findings: Organic and Inorganic PERLs Detected

Evaluation of four commercially available purification resins revealed the presence of multiple extractables:

Organic compounds: Detected by HRMS and GC-MS, representing base matrix components, ligands, synthesis residuals, or degradation products

Inorganic species: Detected by ICP-MS, including elemental impurities potentially from synthesis catalysts, stabilizers, or base matrix materials

The specific findings validate the concern: commercially available purification resins, despite their critical role in removing impurities, do themselves contain extractable compounds.

From Extractables to Leachables Monitoring

The extractables profile serves a critical forward-looking function. This list of detected compounds becomes a monitoring target list for leachables evaluation:

• During process development: Monitor for these extractables in process streams after chromatography steps
• In drug substance: Test purified protein batches for presence of identified PERLs
• In drug product: Include these compounds in final product leachables monitoring programs
• Under stability: Assess whether PERL levels change during product storage

This targeted monitoring is far more efficient than untargeted screening alone. While comprehensive screening should still be performed, knowing specifically which PERLs to look for enables sensitive, quantitative methods for critical compounds.

Proactive Risk Mitigation

Joel emphasizes proactive screening. The alternative, discovering leachables in finished product or during stability studies, creates serious problems:

• Potential product quality issues requiring investigation
• Possible manufacturing holds while impacts are assessed
• Risk of product recalls if safety concerns emerge
• Regulatory scrutiny of manufacturing processes
• Potential revalidation requirements

Proactive extractables profiling shifts the timeline from reactive problem-solving to proactive risk management. Understanding potential PERLs during equipment selection and process development enables:

• Informed supplier selection: Comparing extractables profiles between resin vendors
• Process optimization: Adjusting conditions to minimize leaching of concerning compounds
• Specification development: Setting appropriate limits for PERLs in process intermediates and final products
• Method development: Creating targeted analytical methods for critical PERLs before routine manufacturing begins
• Control strategy design: Incorporating PERL monitoring into overall process control strategy

This proactive approach aligns with Quality by Design principles and ICH Q11 guidance on drug substance development.

Beyond Purification Resins

While Joel’s work focused on purification resins, the PERLs concept extends to all process equipment contacting drug substance or product:

• Filters and filtration membranes
• Tubing and gaskets
• Storage vessels and bags
• Mixing equipment
• Pumps and valves
• Sensors and probes

Each represents a potential source of process-related leachables deserving systematic evaluation. Comprehensive E&L programs should address the entire manufacturing train, not just primary packaging.

Expert PERLs Evaluation and E&L Profiling

Resolian’s extractables and leachables team brings comprehensive capabilities for manufacturing process impurity evaluation:

• Multi-technique analytical platforms: HRMS, GC-MS, ICP-MS
• Worst-case extraction protocol development
• Both organic and inorganic extractables profiling
• Experience across biopharmaceutical manufacturing equipment
• Targeted leachables monitoring method development
• Risk assessment and control strategy support
• Regulatory guidance on PERLs evaluation

Whether you’re evaluating new manufacturing equipment, investigating unexpected impurities, or developing comprehensive E&L programs that include process equipment, we bring the multi-technique expertise and systematic approach that PERLs evaluation demands.