DEEPAK K. SHARMA,1 CLARK MERCHANT,1 PETER OMA,1 DAVE KING1
1 ProteinSimple Technologies Inc., 115 Terence Matthews Cr., Ottawa, Ontario, Canada, K2M2B2
Purpose:
The need to monitor, measure and control sub-visible proteinaceous particulates in bio-pharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone microdroplets, air bubbles and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-Flow Imaging™ (MFI) has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present.
Methods:
The focus of current study was to investigate the accuracy of MFI to size and count particles in general and proteinaceous particles in particular, in high concentration opalescent protein solutions. Two different protocols were developed to generate proteinous particles of a mAb which were then re-suspended in opalescent concentrated mAb solutions to investigate precision and linearity of detection by MFI or to access accuracy by comparison to light obscuration (LO) and manual microscopy. In another such study MFI was used for the identification and differentiation of protein aggregates, silicone oil, and air bubbles in protein formulations.
Results:
Results showed that MFI can detect proteinaceous particles, even in the context of opalescent formulations. The sizing and concentration accuracy of MFI for polystyrene and glass particles, representing different particle refractive index and transparency characteristics, was unaffected by the opalescence of the medium. MFI also demonstrated a high degree of linearity for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (~20 to 160000 particles/mL). These particles were significantly underestimated by the LO and manual microscopic technique, particularly those in the 2-10 µm size range. Through appropriate morphological filter development, results show successful differentiation of over 94% between protein aggregates, air bubbles and silicone oil in a PBS buffer solution for particles within the USP <788> size limits.
Conclusions:
MFI with automated particle classification is an emerging technology that can play a useful role in resolving and independently characterizing mixed particle populations in biopharmaceutical formulations.
PRESENTED AT:
2010 Workshop on Protein Aggregation and Immunogenicity, Breckenridge CO, USA
2010 ProteinSimple_Subvible Particles in High Conc mAb and Silicone classification.pdf
2010 ProteinSimple_Subvisible Particles in High Conc