Hollow Fiber Flow-Field-Flow Fractionation (HF5) Technology

Hollow fiber flow-field-flow fractionation (HF5), the microvolume variant of AF4, has emerged as a soft size-separation technique to determine the size and number of aggregates and particles in protein preparations, and it has been demonstrated to be a successful alternative to SEC. CD Formulation has extensive experience in developing HF5 methods for a variety of samples. We provide reliable analytical support for protein/peptide drug development and characterization with state-of-the-art HF5 technology.

What is Hollow Fiber Flow-Field-Flow Fractionation (HF5)?

HF5 is a technique for separating and analyzing particles such as polymers, proteins, and nanoparticles based on their size and shape. Compared to AF4, this variant generally has higher or comparable efficiency and higher detectability, i.e. lower dilution of the sample during the separation process. In addition, the technique is highly selective for analytes of very high molar mass and requires only small amounts of sample.

Hollow Fiber Flow-Field-Flow Fractionation (HF5) Principle

HF5 is based on the separation of protein/peptide particles or molecules based on their size, shape, and density.

In this technique, separation is achieved in a hollow capillary channel consisting of a tubular membrane with porous walls. The combination of laminar flow of the mobile phase and orthogonal flow (cross flow) through the membrane determines the separation. The sample passes through the hollow fiber membrane which separates the particles based on their size, which is then further separated based on their diffusivity in the field flow system. Smaller particles migrate closer to the fiber walls, while larger particles remain closer to the center of the fiber, resulting in high-resolution separation of particles and the ability to analyze complex samples with a wide range of particle sizes.

Fig. 1 Schematic of HF5; separation principle and protocol workflow. (Tanase M, et al., 2015)

Our Services Related to Hollow Fiber Flow-Field-Flow Fractionation (HF5)

Benefiting from decades of experience in supporting protein/peptide biopharmaceutical development and manufacturing with HF5 technology, our team of highly qualified experts can provide a range of HF5-related services to accelerate the implementation and success of your project.

Our HF5 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).

Our HF5 technology uses a tubular separation channel to minimize sample interactions, resulting in higher sensitivity and higher separation efficiency. In our protein/peptide analysis laboratory, our scientists use HF5 to study minute amounts of synthetic polymers encapsulating proteins/peptides, as well as encapsulation and release studies of very valuable and expensive proteins or peptides.

Utilizing cutting-edge HF5 technology, we provide the following HF5-related services, including but not limited to:

Separation of Proteins or Peptides of Different Dizes and Molecular Weights Using HF5 Technology

We inject samples containing proteins or peptides of different sizes into a flow system consisting of a hollow fiber channel. The samples of proteins or peptides experience different velocities due to their size and molecular weight, which causes them to separate within the channel.

The separation efficiency of HF5 is highly dependent on flow parameters, including flow rate, carrier fluid composition, and channel geometry. We continuously optimize these parameters according to proteins/peptides particle size and molecular weight, to achieve efficient separation.

Characterization of Small Amounts of Synthetic Polymers Encapsulating Proteins/Peptides Using HF5 Technology

We continuously establish new protocols to deeply characterize polymer delivery vehicles to understand structural transformations that are dependent on loading or modification processes.

Exploration of Loading and Release of Proteins or Peptides in Nanoformulations Using HF5 Technology

HF5 is a powerful tool for the characterization and analysis of nanoparticles, including their size, shape, and surface properties.

Our scientists monitor the changes in protein or peptide concentration over time by combining quantitative analysis detection systems to gain in-depth insights into the release mechanism, stability, and bioavailability of loaded protein/peptide molecules to support nanoformulation research.

Our Equipment

In HF5 analysis, we use multiple detection modalities to characterize and correlate protein/peptide biopharmaceuticals in multiple dimensions. These state-of-the-art analytical platforms enable us to correlate quantitatively and qualitatively to gain in-depth insights into the physical and chemical properties of protein/peptide macromolecules, their self-assemblies, and nano-delivery vehicles.

Available online or offline detection systems include:

• Multi-Angle Light Scattering (MALS)
• Dynamic Light Scattering (DLS)
• Differential Refractometer (DR)
• Differential Viscometer (DV)
• UV-Vis Spectrophotometer(UV-Vis)

Advantages of Our Hollow Fiber Flow-Field-Flow Fractionation (HF5) Technology

• High resolution: Our HF5 provides high-resolution separation of complex mixtures based on differences in particle size and shape, including proteins, nanoparticles, and polymers.
• Scalability: Our HF5 can be easily scaled up to handle larger sample volumes, making it suitable for the protein/peptide biopharmaceutical manufacturing stage.
• Reduced sample dilution: Our HF5 requires minimal sample dilution, allowing for a more accurate analysis of concentrated samples.
• Rapid analysis: Our HF5 enables rapid separation and analysis of samples, reducing the time required for analysis compared to other separation techniques.
• Compatibility with sensitive samples: Our HF5 is gentle on samples, making it suitable for analyzing fragile or sensitive materials without causing damage.
• Low running cost: Our HF5 requires minimal consumables and has low running costs, making it a cost-effective separation technique.

Custom Hollow Fiber Flow-Field-Flow Fractionation (HF5) Services

Why Choose Our Hollow Fiber Flow-Field-Flow Fractionation (HF5) Technology?

• We have a team of experts with rich experience in performing HF5 analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using HF5 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 characterizing any possible aggregates and particles for protein/peptide biologics development and manufacturing. Please feel free to contact us to speak with our team of scientists and learn how our HF5 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.