AF4 Services for Biopharmaceutical 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.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is regarded as the gold standard for aggregation analytics. SV-AUC is a first principle method, and in contrast to size exclusion chromatography (SEC), SV-AUC does not rely on a stationary phase.  Furthermore, SV-AUC samples are not diluted by a mobile phase. For these reasons, regulatory bodies like FDA and EMA show steadily increasing interest in SV-AUC results. We therefore advise to always verify SEC results by a first principle method like SV-AUC. For more information about the use of SV-AUC in biopharmaceutical development please follow the link.

Dynamic light scattering is a valuable tool for biopharmaceutical development. It is a fast and efficient screening method for aggregate / particle detection. DLS closes the gap between the range of small soluble aggregates that are detected via size exclusion chromatography (SEC), and particles analyzed by methods such as light obscuration (LO) or micro flow imaging (MFI). Owing to the technique’s high sensitivity towards large species, even small impurities can be detected within the measurement range of0.5 nm to 1 µm. If you want to hear more about the use of DLS in biopharmaceutical development please follow the link.

Size exclusion chromatography (SEC) is the most widely used technique for aggregate determination in biopharmaceutical development. Obvious advantages are the easy handling and the good reproducibility that this method provides. To avoid misleading results, it is essential to develop a method that is not solely based on the observed elution pattern of the sample, but that also characterizes each observed eluted species individually. The state-of-the-art MALLS detector (Wyatt DAWN Heleos II) at ZentriForce Pharma allows us to obtain the molecular weight of all observed species. With this information we can develop a truly feasible SEC method for you. For more 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). 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 Biopharmaceutical 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 comparably small measurement ranges, AF4 methods can cover a measurement range 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 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 to our AF4 theory page.

AF4 as a Tool in Biopharmaceutical Development

We routinely conduct biosimilarity and gene therapy studies and are furthermore experienced in the development of asymmetrical flow field-flow fractionation (AF4) methods for a large variety of samples.  This includes the application of AF4 within the development of new biopharmaceutical APIs. 

AF4 is a valuable tool for verification of SEC results. It can use the same mobile phase but does not depend on a stationary phase. AF4 thus is an exceptionally useful technique to investigate the effect of the stationary phase on SEC results.

AF4 is applicable to establishing screening methods with large measurement ranges, from 1 nm up to 1 μm. Despite the vast size region, high resolution within the oligomeric range is possible. In combination with a multiangle laser light scattering (MALLS) detector, AF4 allows us to quantify as well as characterize different species, for example in regard to their molecular weight).

The AF4Eval program enables us to increase the resolution of AF4 results significantly. AF4Eval allows the application of a diffusion correction to subtract the well-described broadening effects that occur in AF4 measurements.

ZentriForce Pharma AF4 Instrumentation

Combining the Agilent 1260 Infinity II HPLC, Wyatt DAWN Heleos II MALLS detector and a Wyatt Optilab T-rEX RI detector, we  work with cutting edge instrumentation that offer 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 improved application 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. For more detailed information, follow the link.

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. 

SV-AUC is a high resolution method for the determination of soluble aggregates. It can be seen as the gold standard and can be an helpful tool, especially in cases of troubleshooting. Additionally. it is widely used for the verification of size exclusion chromatography methods.  If you want to hear more about the use of SV-AUC in biopharmaceutical development please follow the link.

DLS is a highly sensitive method for the detection particles. It is a fast and effective screening method for formulation development and production optimization. If you want to hear more about the use of DLS in biopharmaceutical development please follow the link.

Size exclusion chromatography is the most common technique for aggregate determination. If the method is developed properly SEC can give you reliable and   Asymmetrical flow field-flow fractonation is an orthogonal method to size exclusion chromatographie (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC). AF4 can be used for a wide range of applications ranging from the detection of soluble aggregates, characterization of species via multi angle laser light scattering (MALLS) and determination of particle size distributions. For more 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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