We continue in our efforts to confirm that the HPLC-gel permeation chromatography (GPC) methodology used by a particular manufacturer of generic Hetastarch (a heterogenous colloidal plasma volume expander) gives correct Mw (weight average molecular weight) and Mn (number average molecular weight) values. The Division of Drug Analysis of CDER has analyzed numerous lots of innovator and generic (not yet approved) Hetastarch by means of this HPLC-GPC methodology and obtained Mw and Mn values for each lot. Previously at CBER we were able to estimate an upper limit of Mn for innovator Hetastarch of <46,700>5 by membrane osmometry; the ability to determine only an upper limit is due to the presence of very small amounts of species that have molecular weights below the cut-off of the semipermeable membrane. The Mn values for innovator Hetastarch measured by this HPLC-GPC method are in accord with the upper limit determined by membrane osmometry; interestingly, those for lots of generic Hetastarch are substantially greater. In order to confirm the Mw values for both Hetastarches measured by HPLC-GPC, we continue our collaboration with Dr. R. Ghirlando of NIH, who is determining Mw by light scattering; scattering data at 514 nm are measured as a function of concentration and angle and are extrapolated to zero concentration and angle by means of a Zimm plot to obtain Mw. In general, the agreement between Mw values determined by HPLC-GPC and light scattering is very good. Currently, we are endeavoring to make small corrections to the light scattering data by taking into consideration the small amount of sodium chloride (and moisture) present in the Hetastarch powders. To this end, we have started to carry out light scattering measurements in water and to determine (dn/dc)_=546 in water also, as opposed to in 154 mM NaCl; furthermore, dn/dc values will be corrected to 514 nm. In general, the ratio Mw/Mn is r1 and permits an assessment of the polydispersity of the material with unity indicating a monodisperse preparation. Both Hetastarches are extremely polydisperse with Mw/Mn >>1. However, an accurate Mn is not only useful as a monitor of heterogeneity but also as an indicator that the HPLC-GPC column configuration is giving resolution sufficient for the determination of true molecular weights. The inability of other Hetastarch manufacturers to determine accurate Mw and Mn values by HPLC-GPC probably relates to insufficient resolution of the columns.

National Institute of Health (NIH)
Food and Drug Administration (FDA)
Intramural Research (Z01)
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