Best practices for purification and characterization of PAMAM dendrimer

Abstract

Poly(amidoamine) (PAMAM) dendrimer are branched polymers with low degrees of heterogeneity. Current synthesis methods, however, result in substantial batch variability. We present our optimized procedure for post-synthesis (and post-market) purification of a generation 5 PAMAM dendrimer by membrane dialysis and demonstrate its effectiveness and limitations using a representative lot of biomedical grade dendrimer. This method successfully removes trailing generation defect structures, thereby reducing the heterogeneity of the material (PDI reduced from 1.04 to 1.02). Optimized analytical techniques to characterize the unpurified and purified dendrimer are also detailed. The efficiency of the purification method is successfully monitored by these analytics and dendrimer parameters that are critical for subsequent modification reactions and biological evaluation (M(n), M(w), PDI, average number of end groups) obtained. To provide better definition of the variability that should be expected between lots of synthesized material, HPLC traces for three additional commercial lots of dendrimer are also presented.

Figure 1

Characterization of PAMAM dendrimer using a GPC based Light Scattering Detector and a Differential Refractive Index Detector. a) Unpurified Lot A Dendrimer. b) Purified Dendrimer (Aliquot 1). The purification method reduces the amount of smaller dendrimer particles (trailing generation defects) that elute at 22.5 minutes.

Figure 2

Normalized HPLC traces at 210nm for Unpurified Lot A dendrimer and Purified Dendrimer Aliquots 1, 2, 3 and 4. Note that Purified Aliquots 1–4 have overlapping traces. Purification by membrane dialysis successfully removes the trailing generation defect structures with elution times of 16.9 min and 17.8 min but does not remove the trailing generation defect structures at 18.5 min or the dimer defects at 19.3min.

Figure 3

Potentiometric titration of the Purified Dendrimer (Aliquot 1).

Figure 4

Normalized HPLC traces at 210nm for Unpurified Dendrimer Lots A, B, C and D as well as Purified Dendrimer Aliquot 4.