Synthesis and purification of the 1F5-SA anti-CD20 immunoconjugate.

(A) Reaction progress. Size exclusion chromatograms of the conjugation reaction of reduced 1F5 with SMCC-functionalized streptavidin at an early time point (∼5 minutes; solid trace) and a late time point (∼45 minutes; dashed trace). Components present are: 8.0 minute, high-molecular-weight products; 8.7 minutes, desired 1:1 and 1:2 1F5-SA conjugate; 9.0 minute, unreacted 1F5; 10.2 minutes, unreacted streptavidin. (B) Composite purification profile of the 1F5-SA anti-CD20 immunoconjugate monitored by sequential HPLC analysis. Size exclusion chromatograms of the conjugation reaction components isolated during the purification process. Dotted trace, high-molecular-weight byproducts eluted with high salt from the cation exchange column; solid trace, purified 1:1 and 1:2 1F5-SA conjugate eluted from the cation exchange column with 90 mM NaCl; dashed trace, unreacted 1F5 from the “flow-through” of the iminobiotin affinity column; dot-dash trace, residual SA from the “flow-through” of the cation exchange column. The figure illustrates the separation of each nondesired component from the desired conjugate (solid trace), and the amount of desired conjugate lost with each undesired component. The only significant unrecoverable loss of product on purification is in the high-molecular-weight byproduct peak (dotted trace).