Formation of protein sub-visible particles during powder grinding of a monoclonal antibody.

ABSTRACT

We have observed an interesting phenomenon in which grinding of freeze-dried monoclonal antibody X (mAb-X) formulation powder resulted to significant protein sub-visible particles (SbVPs) in the reconstituted liquid, which could only be observed by sensitive particle analytical methods such as MFI and DLS. Effects of grinding temperature and the free radical scavengers methionine and 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidin-yloxy free radical (CTPO) on the formation of SbVPs were also evaluated. Free radicals were observed by EPR and the amount of free radicals was correlated to the sample temperature prior to grinding. Formation of SbVPs could be partially inhibited by methionine and CTPO. The amount of SbVPs formed was dependent on the amount of free radicals/sample temperature prior to grinding. At higher temperatures, more free radicals and SbVPs formed. Other than the previously known protein degradation due to high temperature formed during mechanical grinding, we propose an unreported and supplementary mechanism, i.e., the formation of free radicals (i.e., due to break of CO or CS bonds) in the dried state during mechanical grinding, leading to protein particle formation in the reconstituted solution. Our observation suggested that mechanical grinding of protein powder should be avoided or used cautiously (i.e., grinding temperature, strength and time) and the effects on radical and particle formation be fully evaluated.