Slip flow occurs in colloidal crystals made of 470 nm silica spheres that are chemically modified with hydrocarbon, giving enhanced volume flow rates and a narrower distribution of fluid velocities. Bovine serum albumin separates by pressure-driven flow with a zone that is 15-fold narrower than the theoretical limit for Hagen-Poiseuille flow. The zone variance, normalized for separation length, is 15 nm, which is 500-fold smaller than previous reports for pressure-driven protein chromatography. A colloidal crystal is shown to separate a monoclonal antibody from its aggregates in only 40 s, representing a ten-fold increase in speed. Slip flow thus has profound implications for protein chromatography.

Extremely uniform packing of colloidal silica in capillaries is shown. Reversed-phase electrochromatograms of DiI-C12 exhibit plate heights as low as 0.13 µm and a reduced plate height as low as 0.4, using 75 µm i.d. capillaries packed with 330 nm silica particles. The contribution from the A term is 0±20 nm in electrochromatography. The particles are shown to form colloidal crystals inside the capillaries. Optical images show Bragg diffraction, indicative of crystallinity; SEM images show face-centered cubic crystallinity; and the porosity is 0.25±0.01, which is in agreement with that for face-centered cubic crystals. The capillaries are fritless, and 100 µm i.d. capillaries packed with silica colloidal crystals withstand pressures of at least 12,400 psi.

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.