High-Performance Size-Exclusion Chromatography

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High-Performance Size-Exclusion Chromatography

Method Introduction

High-performance size-exclusion chromatography (HP-SEC) is a well-established, robust, and highly versatile technique. In HP-SEC, the sample is injected onto a column constantly flushed with mobile phase. The chromatography column contains fine and porous beads composed of dextran, agarose, silica, or polyacrylamide (the stationary phase). The beads allow small species to migrate into their pores, increasing their retention inside the column, while larger species migrate with the mobile phase without entering the bead pores. Thus, larger species reach the column end faster than smaller species. After separation, the various species are detected using the following detectors:

  • Ultraviolet (UV) absorption
  • Fluorescence
  • Refractive index (RI)
  • Multi-angle laser light scattering (MALLS)
  • Charged aerosol detection (CAD)

HP-SEC allows the sizing, quantification, and molecular weight determination of fragments, monomers, and aggregates. The size range of HP-SEC is defined by the column’s pore size and the method set-up (e.g., mobile phase, flow settings, and column dimensions).

Applications

HP-SEC is commonly employed in all stages of biopharmaceutical research and development. It is highly valuable during formulation development, stability, and forced degradation studies but is most frequently used for batch-release testing.

Compared to these orthogonal techniques, HP-SEC generally offers a higher resolution and lower limit of detection:

  • Asymmetric flow field flow fractionation (AF4) offers an increased upper size limit and reducing the bias due to column matrix interactions, allowing for the analysis of larger aggregates and particles. However, method development may be more elaborate.
  • Analytical Ultracentrifugation (AUC) characterizes samples in their native buffer while mitigating dilution effects. As a matrix-free separation technique, AUC is very suitable for verifying HP-SEC results. The modern instruments operated by Coriolis can even reach the throughput of HP-SEC sequences.

Verification of HP-SEC data with orthogonal techniques, such as AUC and AF4, is often recommended and can be performed by Coriolis during method development or on existing methods.

Quality and Biosafety Level

We provide all our analytical services with the highest quality standards. Experienced scientists carry out each project, and a scientific reviewer comprehensively checks every report or data presentation.

We offer this technology with the following quality and biosafety levels:

Up to GMP Level

This method is available under GMP in dedicated labs with qualified equipment and full QA involvement.

Up to biosafety level 2

This method can be applied to nucleic acids, viruses, cells, viral vectors, including lentiviruses, and more.

High-Performance Size-Exclusion Chromatography (HP-SEC) Frequently Asked Questions (FAQs)

  • HP-SEC is an analytical technique used to separate and quantify molecular species based on size. It is widely used to analyze protein monomers, fragments, and aggregates by measuring their elution times through a porous stationary phase.

  • A sample is injected into a chromatography column filled with porous beads. Larger molecules elute faster because they cannot enter the bead pores, while smaller species are retained longer. Detection systems, such as UV, MALLS, or refractive index, track each species as it exits the column.

  • HP-SEC is commonly used to analyze biopharmaceutical proteins, peptides, viral vectors, nucleic acids, and nanoparticles. It is especially effective for sizing and quantifying monomers, fragments, and aggregates during development, manufacturing, and release testing.

  • HP-SEC is applicable at all stages, from early formulation development to final product batch release. It supports stability studies, forced degradation studies, and comparability assessments during scale-up or process changes.

  • HP-SEC offers high resolution, low detection limits, and established method robustness. Compared to orthogonal techniques like AUC or AF4, it offers faster analysis and greater routine applicability, although AUC may provide superior accuracy in native buffers and AF4 a higher upper size limit.

  • Yes. Coriolis offers HP-SEC testing up to GMP level, using qualified instruments in controlled labs with full QA involvement, making it suitable for regulated programs and commercial release testing.

  • Coriolis employs multiple detectors with HP-SEC, including:

    • Ultraviolet (UV) absorption
    • Fluorescence detection
    • Refractive Index (RI)
    • Multi-Angle Laser Light Scattering (MALLS)
    • Charged Aerosol Detection (CAD).

  • Yes. Coriolis recommends and offers verification of HP-SEC results using orthogonal methods such as analytical ultracentrifugation (AUC) or asymmetric flow field-flow fractionation (AF4) to enhance data robustness and regulatory confidence.

  • HP-SEC may not be ideal for very large aggregates or particles that exceed the column’s size exclusion limit. The results can also be affected by dilution effects, interactions with the column matrix or changes induced by the mobile phase. These issues can often be addressed through complementary techniques like AUC or AF4.

  • HP-SEC services at Coriolis are available up to biosafety level 2 (BSL-2), allowing analysis of complex samples such as viruses, viral vectors (including lentivirus) and cell-based materials.

Analytical Method Development, Qualification and Validation

For common sample types, we can often apply standardized methods with little setup effort. However, when needed, our experienced analytical experts create or optimize custom methods tailored to your active pharmaceutical ingredient, product type and development phase.

Method Development

Our method development approach tailors sample preparation, method settings and data analysis to the needs of your project and sample.

We include representative samples and, where available, suitable reference standards and stressed/degraded materials, allowing our analytical scientists to design a highly suitable, stability-indicating, robust and repeatable method. Upon request, we will compile a detailed description of the method for your records.

Method Qualification

Method qualification is the initial assessment of an analytical procedure’s performance to show its suitability for its intended purpose.

During method qualification, our analytical scientists perform documented testing demonstrating that the analytical procedure meets criteria in several categories. Criteria may include factors such as repeatability, specificity and robustness. We compile a qualification plan and report, including all relevant data.

Method Validation

Under GMP conditions, method validation confirms that an analytical procedure’s performance suits its intended purpose. Depending on the method’s scope, a broad range of method characteristics, such as specificity, accuracy, precision, limit of detection/limit of quantification (LOD/LOQ), linearity and range, is considered.

During method validation, our analytical scientists perform documented testing demonstrating that the analytical procedure consistently produces a result that meets the predetermined acceptance criteria. We compile a validation plan and report that includes all relevant data.

Depending on the development phase, a fit-for-purpose validation approach can be offered, adjusting the validation required efforts in a phase-appropriate way to meet the method’s needs.

Method Verification

Compendial method verification confirms that a compendial method (e.g., from Ph. Eur. or USP) is suitable and reliable for its intended purpose under the specific conditions of the laboratory.

Unlike full method validation, compendial method verification is often considered a partial validation since the method has already undergone extensive testing and validation during its inclusion in the compendium. The extent of method verification depends on the type of method.

During method verification, our analytical scientists perform documented testing demonstrating that the developed analytical method performs adequately for the specific product or matrix being tested and within the specific laboratory where the method will be employed.

Talk to Our Experts or Request a Quote

Our expert team is ready to answer your questions and guide you to the services best suited to your program’s modality, stage and challenge. If your needs are well-defined, we’ll begin the quotation process.

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