The Effect of Polysorbate
Figure 7 shows the effect of including a low amount (0.004% by weight) of Tween 80 (polysorbate 80) on the particle counts
in a particular IgG liquid formulation. Samples were stored at 4 °C for three months and then analyzed for particulates. These
IgG preparations were either derived from a hybridoma cell line or a Chinese hamster ovary (CHO) cell line. In the absence
of Tween, the hybridoma-derived material had higher particle counts compared to the CHO-derived material. This difference
may be a result of the inherent nature of the protein molecule or differences between the two processes. A size range of 2,
5, 7.5, 10, 20, and 25 μm is shown at 10 and 20 mg/mL. As evident from the graph, the 2 μm counts are orders of magnitude
higher than the other size range and should be an important consideration in the particle analysis.10
The Effect of pH
Figure 8 shows the effect of varying pH on particulate counts for 10-, 20-, and 25-μm particle size. Samples were formulated
in a poly buffer system with a common excipient to maintain osmolality. Samples were then stressed over 24 h using a tumbling
apparatus. The tumbling action was used to represent agitation stress that may be experienced during the transportation of
drug product. This particular antibody is more stable in the acidic pH range from 5 to 6. At neutral and basic pH, however,
the particle counts are significantly increased. The exact reason for this particle increase as a function of pH is not known,
but it may be related to changes in the surface charge distribution of the molecule as the pH is increased from 5.0 to 7.5,
causing the protein to become less soluble. It was also noted that formulations containing higher particulate counts showed
increased dimer levels by size exclusion chromatography (data not shown).
Detecting Particles with Flow Imaging Technology
Recently, flow imaging technology has emerged as an orthogonal technique to measure subvisible particles, in addition to light
obscuration–based techniques.30 In this setup, samples are made to flow through a microfluidic cell, and digital images of suspended particles are captured.
The images are then analyzed by the software to count particles and estimate their size. In addition to being an orthogonal
technique to light obscuration, flow imaging also has the advantage of making it possible to view the particle in question.
The image and the aspect ratio (ratio of longer to shorter dimension) helps differentiate if the particle is an aggregate
or silicon-oil droplet, some other foreign particle, or even an air bubble.
Figure 9 shows particulate analysis using microflow imaging (MFI) of a different IgG2 monoclonal antibody. Particulation behavior
of this molecule stored in a glass prefilled syringe was compared to a glass vial, in a formulation that lacked polysorbate.
Data are plotted as total particle counts for a variety of particle ranges (from 2 to >125 μm). Under these conditions, the
prefilled syringe produced significantly more particles than the glass vial, across the different size ranges (up to >50 μm).
This was most likely caused by the phenomenon of silicon-oil–induced particulation.31
Characterizing and controlling particulates through a rational formulation screening process is an important part of protein
drug development. Careful analysis of particle generation through downstream processing, storage, and transportation should
be an important consideration in drug development. Furthermore, particle detection and quantification using advanced techniques
has become an integral part of biopharmaceutical development.