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www.postnova.com·info@postnova.com27.11.03-12：0903-03-01-01 NovaSheet Technology： AF4-UV/Vis Application： Characterisation of Recombinant Antibodies- Study on Conformation， Aggregation and Fragmentation Recombinant Antibodies arewidelyused in modern drugformulations. Asymmetric Flow FFF (AF4) is the premier technique forthe characterisation of this kind of antibodies. The main reason for thisis， that AF4 can characterise not only the antibody itself， but also it’sfragments and aggregates. Furthermore even small differences in theconformation and the generalheterogeneity of sample can bedetermined. Fig. 1： Characterization of a Antibody with Aggregates and Fragments. Fig. 1 shows that using the postnova AF2000 system not only theantibody itself， but also two fragments and the higher aggregates couldbe separated and characterised.Also the heterogeneity of the antibodyitself can be shown by using this AF4 system， as the peak shape showssome broadening at higher retention time， indicating that the antibodyconformation has changed in that region. Fig. 2： Effect of different Processing Temperatures on the Antibody. Processing Temperature 30℃ Fig. 2 shows the dependence of the antibody from different processingtemperatures. Clearly， the results show， that no fragments andaggregates can be detected when the antibody is processed at atemperature of 30 ℃. The measurement shows additionally theheterogeneity of the antibody， as the peak shape is not symmetric butshows a broadening to higher retention times， indicating a differentconformation of the antibody in this region. Processing Temperature 30-40°C Already at a slightly elevated processing temperature of 30-40°C theantibody shows huge changes in its constitution. Two fragment peakscan be detected in the sample. These fragmentation process is causedby the increased temperature. The main antibody peak stays more orless the same， but only shows a slightly increased broadening to higherelution times. That shows that these antibody species with an differentconformation are also increasing at 30-40C processing temperature.On top of that， some amount of aggregates is generated by the highertemperature， as the small peak eluting right after the main peak of theantibody indicates. Processing Temperature 40°C Even more changes in the constitution of the antibody sample occurewhen the processing temperature is increased to 40°C. Please note，that the injection volume was smaller than in the measurements donbefore. The main antibody peak is changing dramatically， as itdecreases in size a lot and also the antibody fraction with the differentconformation is being terminated nearly completely. The fractogramshows an antibody peak which is much more symmetric than the peaksdiscovered before and which is shifted to earlier elution times. Thisindicated that the conformation of the antibody changed in such a way，that the size is now smaller and the diffusion coefficient is now higherthan before. Also aggregates of the main antibody peak can bedetected in this sample. But it looks like that the main sample which ismissing in the main peak， is deteriorated and has formed the increasedh ramount of fragments which can be found in this sample. So theincreased processing temperate is primarily causing a higher amount offragments than an higher amount of aggregates. Why use AF2000 for Antibody Characterisation? Highh resolution separationn of antibodyconformation， aggregates and fragments. Fast， gentle and nearly interaction free separationwithout the use of a stationary phase. Fraction collection and easy direct coupling with further analytical techniques as MALDI， ELISA， etc.Different antibody conformations can be detected. For further information about recombinantantibody characterisation by FFF， please do nothesitate to contact us at： info@postnova.com.