DEEPAK K. SHARMA,1 CLARK MERCHANT,1 DAVE KING,1 PETER OMA1
1ProteinSimple Technologies Inc., 115 Terence Matthews Cr., Ottawa, Ontario, Canada, K2M2B2
Purpose:
Recent publications and commentaries have emphasized the need to monitor and control populations of sub-visible, proteinaceous particles in bio-pharmaceutical formulations. Some of these particles 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. The need has therefore been identified for new analytical methods which can accurately measure these particle types. The purpose of this research is to demonstrate the suitability of Micro-Flow Imaging (MFI) for addressing these analytical requirements, through the development of a method validation protocol and application upon a model protein formulation.
Methods:
MFI is a ‘Flow Microscopy’ particle characterization technique which measures proteinaceous particles through automated sample introduction, digital image acquisition and image analysis. In this research, a protocol for method validation was developed using the DPA 4100 imaging flow microscopy system and the Series B sample introduction system. The validation protocol was subsequently applied to a stable suspension of sub-visible proteinaceous particles formed using IgG1 immunoglobulin diluted into PBS buffer, and freeze/thaw cycling to induce aggregation.
Results:
This paper describes the method development requirements for protein particle characterization using MFI. The method addresses the issues and implications of transparency, fragility and instability of these particle types. It determines acceptable measurements for a particular drug while considering sources of measurement variation including multiple sample aliquots, multiple drug samples from different sites, and multiple instruments. The need for additional validation methods beyond the use of polystyrene bead size and concentration standards is discussed. The potential use of software filters to remove sub-populations of undesired particle types (i.e. bubbles, silicone oil micro-droplets) to gain a more accurate measurement of the target population and/or to establish additional control limits for specific particle types of concern is described.
Conclusions:
The unique analytical requirements required to successfully count and size sub-visible particles present within protein formulations has been considered, and a protocol for method validation specific to the Micro-Flow Imaging (MFI) analysis technique has been developed and successfully applied upon a model protein containing a stable suspension of protein aggregates.
PRESENTED AT:
2009 AAPS National Biotechnology Conference, Seattle WA, USA
2009 Colorado Protein Stability Conference, Breckenridge CO, USA
2009 CHI PepTalk, San Diego CA, USA
