AF4 Services for Biosimilarity Studies

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.

Within biosimiliarity studies, SV-AUC is commonly applied to characterize the API and quantify aggregates and fragments. SV-AUC is a first principle method and delivers information such as relative concentrations, sedimentation coefficients, molecular weights and shape information (frictional ratio) within a single measurement. While its orthogonal technique SEC might show unexpected (and falsely interpreted) results due to interactions of sample and column material, SV-AUC is not prone to change the sample composition. For this reason, regulatory bodies like FDA and EMA show steadily increasing interest in SV-AUC results.  Please follow the link for more information.

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. This is possible by coupling a multiangle laser light scattering detector (MALLS) with the HPLC instrument. With this information we are able to strengthen your claim of biosimiliarity. For more information please follow the link.

Dynamic light scattering is a crucial part of every biosimilarity study. It is a fast and efficient screening method for aggregate and 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 (0.5 nm – 1 µm). If you need 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). 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 Biosimilarity Studies 

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 size 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 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, follow the link.

AF4 as Tool in Biosimilarity Studies

Our scientists have already supported a wide range of biosimiliarity studies. We are happy to assist you in the development of your biosimilar as well. 

AF4 is a valuable tool for the verification of SEC results. It can be used with the same mobile phase but does not depend on a stationary phase. Therefore, it is suitable to back up your SEC methods.

AF4 is suited to establish screening methods for large size ranges (1 nm – 1 μm). Despite the considerable size spectrum, high resolution within the oligomeric range can be achieved. The combination of AF4 with a multiangle laser light scattering (MALLS) detector enables us to not quantify and characterize the different species.

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 Quality System

We are aware that all AF4 measurements are relevant for your regulatory submissions. We have therefore implemented a strict quality system to guarantee high quality and traceability of all AF4 data. A wide range of quality related standard operation procedures (SOPs) in place. Our quality manager ensures that our scientist follow all rules and instructions laid out within each SOP. We invite all (potential) costumers to audit our facility, our quality system and the data we collect for them.  Please follow the link for a detailed description of our quality system.

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. Please follow the link for more details.

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.

SV-AUC is an important part of a complete biosimilarity study. The regulatory bodies like FDA and EMA a more and more insistent on  seeing SV-AUC data for the submission. ZentriForce Pharma is your partner for the development of a robust and specific SV-AUC method. Please follow the link for more information.

Size exclusion chromatography is the most common technique for aggregate determination. However, most labs only collect concentration signals without characterization of the detected species. With our SEC-MALLS instrument we are not only able to assure that our SEC methods are specific. We also can measure the identity of the detected species to backup your biosimilarity claim.  For more information please follow the link.

Dynamic light scattering is known as a fast and efficient screening method for aggregate / particle detection. Therefore, it is widely used within biosimilarity studies to close the gap between results gained in the range of small soluble aggregates via size exclusion chromatography (SEC) and particle methods such as light obscuration (LO) and micro flow imaging (MFI). Due to the high sensitivity toward large species, even small impurities can be detected within the measurement range (0.5 nm – 1 µm). If you need 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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