Pembrolizumab microgravity crystallization experimentation

Abstract

Crystallization processes have been widely used in the pharmaceutical industry for the manufacture, storage, and delivery of small-molecule and small protein therapeutics. However, the identification of crystallization processes for biologics, particularly monoclonal antibodies, has been prohibitive due to the size and the flexibility of their overall structure. There remains a challenge and an opportunity to utilize the benefits of crystallization of biologics. The research laboratories of Merck Sharp & Dome Corp. (MSD) in collaboration with the International Space Station (ISS) National Laboratory performed crystallization experiments with pembrolizumab (Keytruda^®) on the SpaceX-Commercial Resupply Services-10 mission to the ISS. By leveraging microgravity effects such as reduced sedimentation and minimal convection currents, conditions producing crystalline suspensions of homogeneous monomodal particle size distribution (39 μm) in high yield were identified. In contrast, the control ground experiments produced crystalline suspensions with a heterogeneous bimodal distribution of 13 and 102 μm particles. In addition, the flight crystalline suspensions were less viscous and sedimented more uniformly than the comparable ground-based crystalline suspensions. These results have been applied to the production of crystalline suspensions on earth, using rotational mixers to reduce sedimentation and temperature gradients to induce and control crystallization. Using these techniques, we have been able to produce uniform crystalline suspensions (1-5 μm) with acceptable viscosity (<12 cP), rheological, and syringeability properties suitable for the preparation of an injectable formulation. The results of these studies may help widen the drug delivery options to improve the safety, adherence, and quality of life for patients and caregivers.

Keywords: Biochemistry; Biophysical chemistry.

Fig. 1

HH-PCF hardware: a 1 ml polysulfone bottle with aluminum cap. b Base plate with one tower of 7 × 1 ml polysulfone bottles with aluminum caps and orange gasket for sealing. c Outer aluminum cover, which covers the base plate.

Fig. 2

Experiment overview: Illustration of the timing and sequence of the overall experiment process from lab setup to recovery of the stowage bag. Center photo insert; Astronaut Thomas Pesquet (European Space Agency) removing the HH-PCF assemblies for return in stowage bag. Falcon-9 launch image on right is permissible to use within the public domain, courtesy of SpaceX. The center photo insert was obtained by written informed consent from Thomas Pesquet. The left image of the International Space Station is permissible within the public domain, courtesy of NASA.

Fig. 3

SONICC analyses: visible, UV two-photon excited fluorescence and second-harmonic generation images for the ground and flight experiments. Particles were confirmed to be proteinous based on positive UV. There are 22 trytophans in pembrolizumab and their crystals give a strong UV signal. The pembrolizumab crystals are chiral and therefore give a strong SHG signal. Although the shown images are at slightly different focal planes, all the observed visible particles were UV and SHG positive. The upper panel is a representative sample of bottle contents from a ground experiment by visible (×200), UV, and SHG imaging. The bottom panel: the visible, UV, and SHG image from a representative flight experiment.

Fig. 4

Comparison of ground (left column) and flight (right column) bottles by particle size analyses of same bottles (a). The data are presented graphically as q%, which is the density distribution at a size vs. the particle size diameter in micrometers. Below is a table of the distribution D10, D50, and D90. The DX is defined as the diameter where X% of the population lies below this value. The particle size analyses overlays (color coded) from three independent ground and flight experiments are shown in b.

Fig. 5

Competitive binding assay of flight and ground dissolved crystals and complimentary mother liquors. Dissolved crystals contain binding activity >94% relative to reference pembrolizumab (N = 3, 95% CI).