Evaluation of Crystal Zenith Microtiter Plates for High-Throughput Formulation Screening

Abstract

Formulation screening for biotherapeutics can cover a vast array of excipients and stress conditions. These studies consume quantities of limited material and, with higher concentrated therapeutics, more material is needed. Here, we evaluate the use of crystal zenith (CZ) microtiter plates in conjunction with FluoroTec-coated butyl rubber mats as a small-volume, high-throughput system for formulation stability studies. The system was studied for evaporation, edge effects, and stability with comparisons to type 1 glass and CZ vials for multiple antibodies and formulations. Evaporation was minimal at 4°C and could be reduced at elevated temperatures using sealed, mylar bags. Edge effects were not observed until 12 weeks at 40°C. The overall stability ranking as measured by the rate of change in high molecular weight and percent main peak species was comparable across both vials and plates at 4°C and 40°C out to 12 weeks. Product quality attributes as measured by the multi-attribute method were also comparable across all containers for each molecule formulation. A potential difference was measured for subvisible particle analysis, with the plates measuring lower particle counts than the vials. Overall, CZ plates are a viable alternative to traditional vials for small-volume, high-throughput formulation stability screening studies.

Keywords: antibody(s); automation; developability screening; formulation; high throughput technology(s); protein(s); stability.

Figure 1

From left to right, an example of a CZ plate, a CZ vial, and a glass vial.

Figure 2

The percent volume remaining in a CZ plate after storage at 30°C for 6 weeks. The heavy black line surrounds the inner 60 wells.

Figure 3

Evaporation from CZ plates with and without a mylar bag seal at 30°C and 40°C. Volumes are based on the inner 60 wells. Due to high evaporative losses, plates without mylar bags were not measured at the 13-week time point and only the 30°C condition with the mylar bag was measured at 26 weeks *The 40°C condition was measured at 12 weeks, 30°C at 13 weeks.

Figure 4

Evaporation from CZ plates across multiple temperatures and storage lengths. The 30°C and 40°C plates used the mylar bag seal method and report only the inner 60 wells whereas the −70°C, −30°C, and 4°C temperatures do not use the mylar bag and report the percent volume remaining across all wells. *−70°C, 4°C, and 30°C were measured at 13 weeks, −30°C at 14 weeks, and 40°C at 12 weeks.

Figure 5

SEC results for the Fc-fusion protein across the inner 60 wells of a CZ plate in A52ProPl after (a) 4, (c) 8, and (e) 12 weeks and P62NaSuT after (b) 4, (d) 8, and (f) 12 weeks at 40°C. The color scale for (e) has been limited to 8%-12% to better show gradations.

Figure 6

The individual well results for percent main from rCE-SDS: (a), (c), and (e) are A52ProPl at 4, 8, and 12 weeks, respectively, whereas (b), (d), and (f) are P62NaSuT at 4, 8, and 12 weeks, respectively.

Figure 7

Rate of HMW formation in CZ plates, glass vials, and CZ vials over 12 weeks at (a) 4°C and (b) 40°C; n = 5 for the plate, and error bars are the standard error in HMW rate.

Figure 8

Rate of (a) HC or main, (b) MMW, and (c) LMW formation over 12 weeks at 40°C for CZ plates, glass vials, and CZ vials; n = 5 for the plate, and error bars are the standard error in rate.

Figure 9

Cumulative counts per mL greater than or equal to 2 μm particle diameter in (a) the CZ plate (n = 2), CZ vial (n = 3), and glass vial (n = 3) after 12 weeks on stability for the Fc-fusion protein formulated in P62ProT and P62ProPl and (b) the CZ plate, CZ vial, and glass vial for mAb 3 over 7 days at room temperature (n = 3).