Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway.

Rosmarinic acid (RA) has gained attraction in bioprocessing as a media supplement to improve cellular proliferation and protein production. Here, we observe up to a two-fold increase in antibody production with RA-supplementation, and a concentration-dependent effect of RA on cell proliferation for fed-batch Chinese hamster ovary (CHO) cell cultures. Contrary to previously reported antioxidant activity, RA increased the reactive oxygen species (ROS) levels, stimulated endoplasmic reticulum (ER) stress, activated the unfolded protein response (UPR), and elicited DNA damage. Despite such stressful events, RA appeared to maintained cell health via mammalian target of rapamycin (mTOR) pathway activation; both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) were stimulated in RA-supplemented cultures. By reversing such mTOR pathway activity through either chemical inhibitor addition or siRNA knockdown of genes regulating the mTORC1 and mTORC2 complexes, antibody production, UPR signaling, and stress-induced DNA damage were reduced. Further, the proliferative effect of RA appeared to be regulated selectively by mTORC2 activation and have reproduced this observation by using the mTORC2 stimulator SC-79. Analogously, knockdown of mTORC2 strongly reduced X-box binding protein 1 (XBP1) splicing, which would be expected to reduce antibody folding and secretion, sugging that reduced mTORC2 would correlate with reduced antibody levels. The crosstalk between mTOR activation and UPR upregulation may thus be related directly to the enhanced productivity. Our results show the importance of the mTOR and UPR pathways in increasing antibody productivity, and suggest that RA supplementation may obviate the need for labor-intensive genetic engineering by directly activating pathways favorable to cell culture performance.

Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

Recombinant proteins represent almost half of the top selling therapeutics-with over a hundred billion dollars in global sales-and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N-glycan micro- and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP-GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.

Progress toward rapid, at-line N-glycosylation detection and control for recombinant protein expression.

Proteins continue to represent a large fraction of the therapeutics market, reaching over a hundred billion dollars in market size globally. One key feature of protein modification that can affect both structure and function is the addition of glycosylation following protein folding, leading to regulatory requirements for the accurate assessment of protein attributes, including glycan structures. The non-template-driven, innately heterogeneous N-glycosylation process thus requires accurate detection to robustly generate protein therapies. A challenge exists in the timely detection of protein glycosylation without labor-intensive manipulation. In this article, we discuss progress toward N-glycoprotein control, focusing on novel control strategies and the advancement of rapid, high-throughput analysis methods.

Moving Protein PEGylation from an Art to a Data Science.

PEGylation is a well-established and clinically proven half-life extension strategy for protein delivery. Protein modification with amine-reactive poly(ethylene glycol) (PEG) generates heterogeneous and complex bioconjugate mixtures, often composed of several PEG positional isomers with varied therapeutic efficacy. Laborious and costly experiments for reaction optimization and purification are needed to generate a therapeutically useful PEG conjugate. Kinetic models which accurately predict the outcome of so-called "random" PEGylation reactions provide an opportunity to bypass extensive wet lab experimentation and streamline the bioconjugation process. In this study, we propose a protein tertiary structure-dependent reactivity model that describes the rate of protein-amine PEGylation and introduces "PEG chain coverage" as a tangible metric to assess the shielding effect of PEG chains. This structure-dependent reactivity model was implemented into three models (linear, structure-based, and machine-learned) to gain insight into how protein-specific molecular descriptors (exposed surface areas, pKa, and surface charge) impacted amine reactivity at each site. Linear and machine-learned models demonstrated over 75% prediction accuracy with butylcholinesterase. Model validation with Somavert, PEGASYS, and phenylalanine ammonia lyase showed good correlation between predicted and experimentally determined degrees of modification. Our structure-dependent reactivity model was also able to simulate PEGylation progress curves and estimate "PEGmer" distribution with accurate predictions across different proteins, PEG linker chemistry, and PEG molecular weights. Moreover, in-depth analysis of these simulated reaction curves highlighted possible PEG conformational transitions (from dumbbell to brush) on the surface of lysozyme, as a function of PEG molecular weight.

Organophosphate detoxification by membrane-engineered red blood cells.

Biotherapeutics have achieved global economic success due to their high specificity towards their drug targets, providing exceptional safety and efficiency. The ongoing shift away from small molecule drugs towards biotherapeutics heightens the need to further improve the pharmacokinetics of these biological drugs. Three pervasive obstacles that limit the therapeutic capacity of biotherapeutics are proteolytic degradation, circulating half-life, and the development of anti-drug antibodies. These challenges can culminate in limited efficiency and consequently warrant the need for higher drug doses and more frequent administration. We have explored the coupling of biotherapeutics to long-lived and biocompatible red blood cells (RBCs) to address limited pharmacokinetics. Butyrylcholinesterase (BChE), for example, provides prophylactic protection against organophosphate nerve agents (OPNAs), yet the short circulation life of the drug requires extraordinary doses. Herein, we report the rapid and tunable chemical engineering of BChE to RBC membranes to create a cell-based delivery system that retains the enzyme activity and enhances stability. In a three-step process that first pre-modifies BChE with a cell-reactive polymer chain, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to the surface of each RBC without diminishing the bioscavenging capacity of the enzyme. Critically, this membrane-engineering approach was cell-tolerated with minimal hemolysis observed. These results provide strong evidence for the ability of engineered RBCs to serve as an enhanced biotherapeutic delivery vehicle. STATEMENT OF SIGNIFICANCE: Organophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme's activity and enhancing its stability.