Asymmetrical Flow Field-Flow Fractionation (AF4) Technology

Asymmetrical flow field-flow fractionation (AF4) technology is an interesting alternative to size exclusion chromatography (SEC) for the determination and quantification of aggregates and particles in protein formulations and nano-carrier analysis, including size distribution, drug encapsulation, and release studies. CD Formulation has extensive experience in developing AF4 methods for a variety of samples. With best-in-class AF4 technology, we provide reliable analytical support for protein/peptide drug development and characterization.

What is Asymmetrical Flow Field-Flow Fractionation (AF4)?

AF4 is a mature fractionation technique that has been recognized as a valuable tool for characterizing the particle size, polydispersity, drug loading, and stability of proteins, peptides, aggregates, and nanomedicines. This technique can provide separation efficiencies comparable to SEC, which can be used as a conventional alternative to SEC for the separation of high molecular weight compounds. However, the separation principle is very different, the AF4 method can cover the spectrum from 1 nm to 1 μm.

Asymmetrical Flow Field-Flow Fractionation Principle

Unlike SEC, the AF4 method has no stationary phase and separation is achieved only by flow in an empty channel, where vertical flow forces are applied.

Analytes of different hydrodynamic sizes migrate in a laminar flow driven by the cross flow and establish a dynamic equilibrium. Smaller particles (with higher diffusion coefficients) move closer to the middle of the AF4 channel, where the longitudinal flow is faster. The velocity gradient within the channel separates molecules and particles according to their hydrodynamic size, resulting in smaller molecules being eluted before larger molecules. This is exactly the opposite of SEC separations, where large molecules are eluted first.

Fig. 1 Principle of asymmetrical flow field-flow fractionation (AF4). (Eskelin K, et al., 2019)

Our Services Related to Flow Field-Flow Fractionation (AF4)

With decades of experience in supporting protein/peptide biopharmaceutical development and manufacturing with AF4 technology, our team of highly qualified experts can provide a range of AF4-related services to accelerate the implementation and success of your project.

Our AF4 technology supports all stages of your protein/peptide drug development and manufacturing - from early research to late-release testing under GMP. It serves as a valuable tool for orthogonal cross-validation of high-performance size exclusion chromatography (HP-SEC), electrophoresis, and analytical ultracentrifugation (AUC).

Utilizing cutting-edge AF4 technology, we provide the following AF4-related services, including but not limited to:

• Protein and peptide separation services.
• Protein/peptide aggregate analysis services.
• Protein/peptide biopharmaceutical similarity study services.
• Protein/peptide biopharmaceutical forced degradation study and real-time stability study services.
• Protein/peptide nanoformulation analysis services.
  a. Determine size distribution and physical properties of nanocarriers
  b. Perform biomaterial characterization to support pre-formulation studies
  c. Perform PEGylation and other polymer shell characterization to support formulation studies
  d. Evaluate excess reagents in formulations.
  e. Perform drug encapsulation and release studies to determine the ease of drug loading.

Our Equipment

We combine a variety of detectors to provide the highest sensitivity, precision, and reproducibility for critical data in AF4 analysis. Common detectors include:

• UV, refractive index, and fluorescence detectors for concentration determination/quantitation detection systems.
• Multi-angle laser light scattering (MALS) detectors for determining molecular weight and size of separated substances.
• DAD and FLD detectors for drug encapsulation and release studies.

Fig. 2 Online and offline detection system for AF4. (CD Formulation)

By combining these online and offline detection systems, we are able to develop and validate a variety of AF4 analysis methods.

Advantages of Our Flow Field-Flow Fractionation (AF4)

• High-resolution separation: AF4 offers a high-resolution separation of particles based on their size and/or molecular weight, allowing for better characterization of complex mixtures.
• Minimal sample preparation: AF4 requires minimal sample preparation, reducing the risk of sample loss or contamination.
• Versatility: AF4 can be used for a wide range of sample types, from nanoparticles to large macromolecules. It is suitable for a variety of applications, including protein analysis, polymer characterization, and nanoparticle sizing.
• Gentle separation conditions: AF4 operates under mild and non-destructive conditions, making it suitable for sensitive samples such as biological molecules. This helps to maintain the integrity of the sample and prevents aggregation or denaturation.
• Continuous and online monitoring: AF4 allows for continuous and real-time monitoring of the separation process, providing valuable insight into the behavior of the sample components. This can help to optimize the separation conditions and improve overall efficiency.
• Compatibility with other analytical techniques: AF4 can be coupled with other analytical techniques, such as UV-Vis spectroscopy, mass spectrometry, and dynamic light scattering, to provide comprehensive characterization of complex samples.

Custom Asymmetrical Flow Field-Flow Fractionation (AF4) Services

Why Choose Our Flow Field-Flow Fractionation (AF4) Technology?

• We have a team of experts with rich experience in performing AF4 analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using AF4 technology to support protein/peptide biopharmaceutical development.
• Our laboratories are equipped with state-of-the-art equipment and technologies to support any type of protein characterization.
• We provide flexible experimental design and customized solutions.
• Fast turnaround time: detailed technical reports are provided within 5-7 days.

Publication

CD Formulation aims to provide a powerful analytical tool for determining and quantifying aggregates and particles in protein formulations and nanocarrier analysis. Please feel free to contact us to speak with our team of scientists and learn how our AF4 technology can support your product development.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.