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During biomanufacturing, several unit operations expose solutions of biologics to multiple stresses, such as hydrodynamic shear forces due to fluid flow and interfacial dilatational stresses due to mechanical agitation or bubble collapse. When these stresses individually act on proteins adsorbed to interfaces, it results in an increase in protein particles in the bulk solution, a phenomenon referred to as interface-induced protein particle formation. However, an understanding of the dominant cause, when multiple stresses are acting simultaneously or sequentially, on interface-induced protein particle formation is limited. In this work, we established a unique set-up using a peristaltic pump and a Langmuir-Pockels trough to study the impact of hydrodynamic shear stress due to pumping and interfacial dilatational stress, on protein particle formation. Our experimental results together demonstrate that for protein solutions subjected to various combinations of stress (i.e., interfacial and hydrodynamic stress in different sequences), surface pressure values during adsorption and when subjected to compression/dilatational stresses, showed no change, suggesting that the interfacial properties of the protein film are not impacted by pumping. The concentration of protein particles is an order of magnitude higher when interfacial dilatational stress is applied at the air-liquid interface, compared to solutions that are only subjected to pumping. Furthermore, the order in which these stresses are applied, have a significant impact on the concentration of protein particles measured in the bulk solution. Together, these studies conclude that for biologics exposed to multiple stresses throughout bioprocessing and manufacturing, exposure to air-liquid interfacial dilatational stress is the predominant mechanism impacting protein particle formation at the interface and in the bulk solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Charge variants of biological products, such as monoclonal antibodies (mAbs), often play an important role in stability and biological activity. Characterization of these charge variants is challenging, however, primarily due to the lack of both efficient and effective isolation methods. In this work, we present a novel use of an established, high productivity continuous chromatography method, known as multi-column counter-current solvent gradient purification (MCSGP), to create an enriched product that can be better utilized for analytical characterization. We demonstrate the principle of this separation method and compare it to traditional batch HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography) methods, using the isolation of charge variants of different mAbs as a case study. In a majority of cases, we are able to show that the MCSGP method is able to provide enhanced purity and quantity of samples when compared to traditional fractionation methods, using the same separation conditions. In one such case, a sample prepared by MCSGP methodology achieved 95% purity in 10 hours of processing time, while those prepared by FPLC and HPLC achieved purities of 78% and 87% in 48 and 300 hours of processing time, respectively. We further evaluate charge variant enrichment strategies using both salt and pH gradients on cation exchange chromatography (CEX) and anion exchange chromatography (AEX) resins, to provide more effective separation and less sample processing following enrichment. As a result, we find that we are able to utilize different gradients to change the enrichment capabilities of certain charged species. Lastly, we summarize the identified mAb charge variants used in this work, and highlight benefits to analytical characterization of charge variants enriched with the continuous chromatography method. The method adds a new option for charge variant enrichment and facilitates analytical characterization of charge variants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure-property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems., (Copyright 2009 Elsevier B.V. All rights reserved.)

After incubation in the presence of tritiated water, incorporation of tritium into cholesterol and into different bile acids was several-fold higher using hepatocytes of cholestyramine-fed rats than that found using hepatocytes of control rats. Labeling of the trihydroxylated cholic and beta-muricholic acids was markedly greater than that of dihydroxycholanoic acid. The total amount of label in all bile acids was 30% or less of that in free cholesterol, in both types of hepatocytes. In combination with the data on bile acids mass production we could calculate the average number (N(a)) of tritium atoms incorporated per molecule of newly-formed bile acid. The experimental values of N(a) for cholic and beta-muricholic acid were compared with values of N(n) or N(o), theoretically predicted if these bile acids were derived entirely from newly made or pre-existent cholesterol, respectively. It was deduced for hepatocytes of cholestyramine-fed rats that the bile acids produced in the first hour of incubation should be totally derived from pre-existent cholesterol, whereas 50% and 25% of the cholic and beta-muricholic acid, respectively, produced during the second and third hours of incubation should be derived from newly synthesized cholesterol. The contribution of newly made cholesterol as substrate for bile acid production was also estimated by using ML-236B. In a concentration of 12 micro M, it depressed cholesterol synthesis by 90% during 1 or 3 hours of incubation of hepatocytes of cholestyramine-fed rats. Mass production of cholic acid was depressed by 25% and that of beta-muricholic acid was not inhibited at all by ML-236B during the first hour of incubation, while they were depressed by 71 and 52%, respectively, during the second plus third hours of incubation. It is concluded that 1) in isolated hepatocytes newly made cholesterol can be a significant substrate for bile acid formation; 2) there are separate cholesterol substrate pools for the productions of cholic or beta-muricholic acid; 3) of the total carbon flux directed into cholesterol synthesis, the major part ends up as free cholesterol and only a minor part as bile acids, even in hepatocytes with a derepressed bile acid production.-Kempen, H. J., M. Vos-van Holstein, and J. de Lange. Bile acids and lipids in isolated rat hepatocytes. II. Source of cholesterol used for bile acid formation, estimated by incorporation of tritium from tritiated water, and by the effect of ML-236B.

Contents of bile acids and lipids, as well as rates of triglyceride synthesis, were determined in isolated hepatocytes from control or cholestyramine-fed rats (denoted below as "control" or "treated" hepatocytes, respectively). During a 3-hr incubation period, total bile acid production was markedly higher in "treated" cells than in "control" cells. With both kinds of cells a marked fall in production rate occurred after the first hour of incubation. Newly produced bile acids appeared in the conjugated form with both kinds of hepatocytes. "Control" cells produced only taurine-conjugated, while "treated" cells made both taurine-conjugated and glycine-conjugated bile acids. However, with exogenous taurine (0.5 mM), the latter cells also produced taurine-conjugated bile acids only. With both kinds of cells, cholic and beta-muricholic acids, but not dihydroxylated bile acids, appeared as newly formed species during the incubation. Addition of dialyzed rat serum to the incubation did not result in a stimulation of bile acid production, with either kind of hepatocytes. "Treated" cells had a slightly higher content of free cholesterol than control cells; contents of other lipids were not different. Fractional release of bile acids and lipids into the medium did not differ between the two kinds of cells. Triglyceride synthesis from added [14C]palmitate (0.5 mM) was 1.8-fold higher in "treated" than in "control" hepatocytes.