AF4 Services for Peptide Development

Related topics:

Asymmetrical flow field-flow fractionation is an orthogonal method to size exclusion chromatography (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC). AF4 is applicable to a wide spectrum of uses, including the detection of soluble aggregates, characterization of species via multiangle laser light scattering (MALLS) and determination of particle size distributions. Follow the link for more information.

Peptides are molecules that are at the lower edge of the size range suitable for classical protein analysis techniques, and too large for established methods for small molecule analysis. Their size thus poses a challenge in analytical method development. Considering this, SV-AUC is an excellent choice for purity assessment of peptide products. Advanced data evaluation approaches are necessary to achieve a resolution that is adequate for this application. We have set up our own computer cluster at ZentriForce Pharma to offer such services, without the need for costumer data to leave our facility. Please follow the link for more information.

Dynamic light scattering is a crucial part of peptide development. It is a fast and efficient screening method for aggregate and particle detection.  In addition, our instrument, the DynaPro®Plate Reader III, allows us to apply static light scattering (SLS) to characterize your peptides. For more information about the use of DLS in peptide development please follow the link.

Size exclusion chromatography method development for peptide samples is a challenging task. The species’ small size results in a low resolution. Nonetheless, relying on our experience, state-of-the-art instrumentation and dedicated team, we can offer cutting edge analytical method development. For more information about the use of SEC-MALLS in peptide development please follow the link.

With the increasing interest in gene therapy products also the interested in AF4 is growing. However, AF4 methods are comparably complex to develop. While most people use AF4 only as separation technique. We are experts in AF4 theory and use the theory to get more information out of the technique (e.g. particle size distributions). If you want to get more information about AF4 theory please follow this link.

The detection of soluble aggregates is a crucial step for a wide range of bio-pharmaceutical products. It can be seen as one of the most important quality parameter, independent of the type of material you want us to analyze for you.  if you have a gene therapy, a proteinaceous or an oligonucleotide based product. We can offer a   We are offering  a wide range of analytical techniques for the determination of soluble aggregates. We can support you with finding the best technique to determine the amount of soluble aggregates as well as their characterization. We also have a specialized web page on this very topic. Please follow the link for more information. 

We are offering a wide range of analytical techniques for the determination of particle size distributions.  The detection of nano particle size distributions (1 nm  – 1 μm) is growing in importance. Especially, since the interest in gene therapy increased. Our scientist are working since more than 10 years on the topic of particle size determination. Please follow the link for more information.

General topics:

ZentriForce Pharma was founded as a service provider that specializes in hydrodynamic techniques for biopharmaceutical development. The focus lies on gene therapy products and biosimilarity studies. Follow the link to our company page for further information.

Please follow this link to our contact page

ZentriForce Pharma implemented a strict quality system to assure the highest tractability of results generated at our facility. We have a wide range of quality related standard operation procedures (SOPS) in place to assure the consistency and quality of our laboratory operations. Please follow the link to a description of our quality system. 

ZentriForce Pharma is your expert in analytical services, with focus on hydrodynamic techniques. Please follow the link to find out about our analytical services. 

ZentriForce Pharma offers a wide range of analytical techniques. Please follow the link to our analytical portfolio. 

ZentriForce Pharma is not merely an expert in analytical development. Our scientist are actively involved in further advances in the theory of hydrodynamic techniques. Follow the link to a detailed description of the theory behind our analytical services, including some of our own research

ZentriForce Pharma is always looking for motivated employees. Check out our career page for further information. 

Asymmetrical Flow Field-Flow Fractionation (AF4) Services for Peptide Development

Asymmetrical flow field-flow fractionation is an orthogonal method to size exclusion chromatography (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC). While the other two techniques are limited to a comparably small measurement range, AF4 methods can cove a size spectrum from 1 nm up to 1 μm.

The separation mechanism of SEC is based on the physical and chemical properties of a stationary phase. Interactions with the stationary phase might have an influence on the measured sample composition. It could, for example, specifically absorb species, split oligomers, or form such. AF4, in contrast, does not rely on a stationary phase and is a first principle method. Therefore, SEC results should be verified by AF4 (or by SV-AUC, which is equally a first principle method). For this reason, regulatory bodies like FDA and EMA show increased interest in AF4 or, alternatively, SV-AUC results to verify SEC data. 

For a more detailed description of AF4 theory please follow the link.

AF4 as a Tool in Peptide development

Although we routinely conduct biosimilarity and gene therapy studies, we are experienced in the development of asymmetrical flow field-flow fractionation (AF4) methods for peptide samples as well.  These experiences also include the application of AF4 within the development of a new peptide-based APIs or within a biosimilarity study (e.g. glatiramer acetate). 

AF4 is valuable tool for the verification of SEC results. It can be used with the same mobile phase and at the same time does not depend on a stationary phase. It offers the opportunity to investigate the effect of the stationary phase of a SEC method on the analyzed sample.

AF4 is suited for screening methods with large measurement ranges and nonetheless gives a high resolution within the oligomeric spectrum. The combination with an multiangle laser light scattering (MALLS) detector enables us to quantify and characterize the different species.

AF4Eval is able to increase data resolution significantly. The program applies a diffusion correction to subtract the well-described broadening effects that occur in AF4 measurements.  Please follow the link for details on AF4 theory. 

AF4 Instrumentation

Combining the Agilent 1260 Infinity II HPLC, Wyatt DAWN Heleos II MALLS detector and a Wyatt Optilab T-rEX RI detector we are working with cutting edge instrumentation that  offers  highest sensitivity, precision and reproducibility.     

Zentriforce Pharma Exclusive AF4 Services

Our scientists are experts in the application of AF4 to a wide range of bio-pharmaceutical products; further, we continuously and actively work on the advancement of AF4 and the utilization of its underlying theory. Starting in 2012, we have developed and improved the AF4Eval program. The software enables us to not only use AF4 underlying theory to determine the particle size distribution of a sample, but also to greatly increase the resolution of the technique by application of a diffusion correction algorithm. We hold the exclusive rights to use the software in a commercial environment but are open to provide our customers with licenses if a method transfer to their facility is requested. The software and its performance is described in detail within the following publication. Follow the link for detailed information,.

As the importance of gene therapeutic products are growing. So is the interest in AF4. AF4 is able to quantify species within a size range of 1 nm up to 1 μm. This makes AF4 the ideal tool for the measurement of adeno-associated viruses, lysosomal formulations, RNA/DNA complexes and even complete viruses. Our scientists have already worked with a wide range of gene therapy products and are able to support you with your very product. We are even are still working on AF4 theory development and are developing new data evaluation approaches. Which allows us to offer AF4 services featuring the AF4Eval program. 

Peptides are a class of molecules whose size makes analysis challenging. However, SV-AUC is an excellent choice for determination of aggregate levels of peptides. Advanced data evaluation approaches are necessary to achieve a resolution that is adequate for this application. Please follow the link for more information.

Dynamic light scattering is a crucial part of peptide development. It is a fast and efficient screening method for aggregate and particle detection.  DLS provides information about particle occurrence. In addition, our instrument, the DynaPro®Plate Reader III, allows us to measure static light scattering (SLS) to characterize your peptides. For more information about the use of DLS in peptide development please follow the link.

Size exclusion chromatography method development for peptide samples is a challenging task. The small size of the species results in a low resolution. Nonetheless, relying on our experience, state-of-the-art instrumentation and dedicated team, we can offer cutting edge analytical method development. For more information about the use of SEC-MALLS in peptide development please follow the link.

The interest of the regulatory authorities get orthogonal results to verity the SEC results is growing. AF4 and AUC are the most prominent orthogonal methods. Additionally, they are bot first principle methods.  AF4 however is a comparably complex technique. Therefore, it is important to find an experienced partner for the development of robust and specif AF4 methods. Our scientist have already worked with a wide range of commercial antibodies and oligonucleotides. Therefore, ZentriForce Pharma is your partner for the development of a robust and specific AF4 method.   If you need further information please follow the link

AF4 is a valuable tool to gain additional, essential information about your product. An major advantage of AF4 is the large particle size range that can be covered in a single measurement (1 nm up to 1μm). Therefore, it can be a useful addition within production optimization or trouble shooting.  A drawback to the AF4 method in the past used to be a comparably low resolution. This problem  was solved by our scientists with the development of the AF4Eval software. For further information please follow the link

With the increasing interest in gene therapy products also the interested in AF4 is growing. However, AF4 methods are comparably complex to develop. While most people use AF4 only as separation technique. We are experts in AF4 theory and use the theory to get more information out of the technique (e.g. particle size distributions). We even developed our own AF4 data evaluation software. Please follow the link to more information about our AF4Eval software. If you want to get more information about AF4 theory please follow this link.

Our scientists are not only applying AF4 we are also working on the advancement of AF4 and its theory. Since 2012 we are working on the development of the AF4Eval program. Since then it was continuously improved. The software enables us to not only to use AF4 theory to determine the particle size distribution but also to tremendously increase the resolution of the technique by applying a diffusion correction algorithm. Please follow the link for more information. 

The detection of soluble aggregates is a crucial step for a wide range of bio-pharmaceutical products. It can be seen as one of the most important quality parameter, independent of the type of material you want us to analyze for you.  if you have a gene therapy, a proteinaceous or an oligonucleotide based product. We can offer a   We are offering  a wide range of analytical techniques for the determination of soluble aggregates. We can support you with finding the best technique to determine the amount of soluble aggregates as well as their characterization. We also have a specialized web page on this very topic. Please follow the link for more information. 

We are offering a wide range of analytical techniques for the determination of particle size distributions.  The detection of nano particle size distributions (1 nm  – 1 μm) is growing in importance. Especially, since the interest in gene therapy increased. Our scientist are working since more than 10 years on the topic of particle size determination. Please follow the link for more information.

Interested in AF4 Services ?

We are more than happy to assist you with our AF4 services.

Asymmetrical Flow Field-Flow Fractionation Specificiation

Main OutputSize distribution (rH or dH); relative concentrations; molecular weight (Mw)
Size range1 – 1000 nm
Additional Outputsradius of gyration (rg) 
Sample Volume4 µL to 100 µL
Measurement typeHPLC vial based
Temperature range 18°C to 40°C
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