(340c) Confocal and Multiphoton Imaging of IgG Gel Layer Formation during Tangential Flow Filtration

The concentration of monoclonal antibodies (mAbs) and other biotherapeutic molecules is the last step in the production of injectable biologics. Cell culture results in a protein product that is purified by a sequence of steps consisting of centrifugation to remove cellular material, protein A capture, and then anion, cation, and hydrophobic interaction chromatography, respectively. Final steps are diafiltration and product concentration by tangential flow filtration (TFF) with an ultrafiltration membrane. TFF concentrates the protein from 5 - 10 mg/mL to 200 - 250 mg/mL. Over this concentration range, viscosity increases with increasing antibody concentration from about 1 to 10² mPa*s, although shear thinning may occur for the proteins at higher concentrations (Apgar et al., 2020). At these conditions, protein gels are hypothesized to form at the membrane’s surface resulting in decreased flux and protein recovery. However, this process is not well understood and has motivated numerous studies on protein accumulation at membrane surfaces.

We report direct measurement of gel formation during concentration of bovine IgG in an operational TFF cell using multiphoton (MP) confocal microscopy to image the TFF membrane channel and to characterize the manner in which protein accumulates within a distance of 1 to 500 µm from the membrane’s surface. The TFF flow cassette is fabricated to enable direct observation of gel formation by MP microscopy and is based on a flow cell design derived from previous work, using single photon confocal microscopy (Zuponcic et al., 2021). Bovine IgG (gamma globulin from blood), consisting principly of IgG1 (150 kD) and IgG2 (163 KD), was used as a surrogate for other types of more-expensive antibodies since relatively large amounts of protein (up to 5 g per run) are required for these experiments and since properties of the bovine IgG are well characterized (Butler, 1969, 1983).

While both MP and single photon confocal microscopy enable observation of protein behavior at operational conditions, MP microscopy utilizes laser light in the infrared range to excite target molecules, causing them to fluoresce at a visible wavelength. Since two photons of light result in a single, high-energy photon, this form of MP is referred to as two photon microscopy (Yoshitake et al, 2016). Unlike conventional confocal microscopy, two photon confocal microscopy minimizes background fluorescence, which makes it more suitable to imaging protein gel layers in contact with high protein concentration solutions. Both single and two photon methods enable imaging of protein within a distance of 1 µm from the membrane’s surface (Zuponcic et al., 2021).

The work reported in this paper is based on a Millipore Ultracel 30 kD (molecular weight cutoff) Pellicon 3 Module, constructed with a regenerated cellulose membrane, coupled with a D type feed screen that is placed on top of or in-between the flat sheet membrane packets. This screen is designed to induce mixing of the viscous feed (i.e., retentate) passing over the membrane through a 1 mm high feed channel. The feed screen is reported to decrease the concentration of stagnant protein at the membrane surface and to enhance mass transfer (Millipore 2003, 2018, Zou et al., 2016)).

We demonstrate that the TFF membrane cassette may be configured and fabricated to enable observation of protein gel layer development on membrane surfaces and along the axial length of an operational ultrafiltration module. This optical flow cassette (OFC) is fabricated from a commercial TFF membrane module through careful machining, employed to expose the feed screen and the membrane underneath. A thin, optically transparent plexiglass cover and rubber gasket are positioned and sealed on top of the feed screen and cassette housing to form an optical channel with a 4 mm optical path length, which is compatible with the 10x objective lens of the confocal microscopes. The overall assembly is constructed in an aluminum housing that fits securely on the microscope stage and may be operated at a maximum of 60 psi (this is also the pressure rating of the commercial TFF module). Detection is based on adding a small aliquot of protein, labeled with Alexaflor 488 with a nominal excitation wavelength of 720 or 830 nm (ThermoFisher Scientific, accessed March 10, 2022). The ratio of labeled to unlabeled protein was 25*10^-4 to 1. Scanning generates Z-stack images of the inside of the cassette, with the images being reconstructed into different views using vendor-provided software.

Two photon confocal imaging shows that gel accumulates within spaces of the feed-screen to a depth of 40 to 60 mm. Unlike single photon confocal microscopy, the background is minimized by directing an infrared laser to the desired point, with emission of two photons occurring from the fluorescent label (Ustione, 2011). By scanning the surface of the membrane at various distances away from the membrane, images are formed that show the presence of protein gel at the membrane surface and within the feed screen. Fluorescence occurs at a specific point because two photons are required to excite the label. This avoids fluorescent background from other components of the flow cell becoming visible and generating a background signal that interferes with observation of the target. Background signals otherwise generated include the plexi cover glass (0.3 mm thick), the void fraction filled with buffer and protein (1 mm thick), and a 0.5 mm thick feed screen.

In this paper we report, for the first time, use of modeling combined with MP microscopy for visualization of the bovine IgG gel layer on a TFF membrane surface at process conditions within an operational cassette where bulk protein concentrations of 250 mg/mL may occur. Together with separate measurements of flux, retentate concentration, transmembrane pressure, and flow rates, these images are helping to inform development of a first-principles, computational model for predicting conditions that maximize protein concentration and recovery. The combination of visualization, process data, and modeling, carried out in a manner not previously possible, sets the stage for understanding mechanisms and impacts of gel layer formation on TFF performance.

References

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