Stefan Kol

One‐step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.

Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.

Predictive glycoengineering of biosimilars using a Markov chain glycosylation model.

Biosimilar drugs must closely resemble the pharmacological attributes of innovator products to ensure safety and efficacy to obtain regulatory approval. Glycosylation is one critical quality attribute that must be matched, but it is inherently difficult to control due to the complexity of its biogenesis. This usually implies that costly and time-consuming experimentation is required for clone identification and optimization of biosimilar glycosylation. Here, a computational method that utilizes a Markov model of glycosylation to predict optimal glycoengineering strategies to obtain a specific glycosylation profile with desired properties is described. The approach uses a genetic algorithm to find the required quantities to perturb glycosylation reaction rates that lead to the best possible match with a given glycosylation profile. Furthermore, the approach can be used to identify cell lines and clones that will require minimal intervention while achieving a glycoprofile that is most similar to the desired profile. Thus, this approach can facilitate biosimilar design by providing computational glycoengineering guidelines that can be generated with a minimal time and cost.

Baicalein Reduces Oxidative Stress in CHO Cell Cultures and Improves Recombinant Antibody Productivity

Oxidative stress that naturally accumulates in the endoplasmic reticulum (ER) as a result of mitochondrial energy metabolism and protein synthesis can disturb the ER function. Because ER have a responsibility on the protein synthesis and quality control of the secreted proteins, ER homeostasis has to be well maintained. When H2 O2 , an oxidative stress inducer, is added to recombinant Chinese hamster ovary (rCHO) cell cultures, it reduced cell growth, monoclonal antibody (mAb) production, and galactosylated form of mAb in a dose-dependent manner. To find an effective antioxidant for rCHO cell cultures, six antioxidants (hydroxyanisole, N-acetylcysteine, baicalein, berberine chloride, kaempferol, and apigenin) with various concentrations are examined individually as chemical additives to rCHO cell cultures producing mAb. Among these antioxidants, baicalein shows the best mAb production performance. Addition of baicalein significantly reduced the expression level of BiP and CHOP along with reduced reactive oxygen species level, suggesting oxidative stress accumulated in the cells can be relieved using baicalein. As a result, addition of baicalein in batch cultures resulted in 1.7-1.8-fold increase in the maximum mAb concentration (MMC), while maintaining the galactosylation of mAb. Likewise, addition of baicalein in fed-batch culture resulted in 1.6-fold increase in the MMC while maintaining the galactosylation of mAb. Taken together, the results obtained here demonstrate that baicalein is an effective antioxidant to increase mAb production in rCHO cells.

Development of a VHH-Based Erythropoietin Quantification Assay

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification of EPO in a high-throughput setting.

The CYP79A1 catalyzed conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime unravelled using an improved method for homology modeling.

The vast diversity and membrane-bound nature of plant P450s makes it challenging to study the structural characteristics of this class of enzymes especially with respect to accurate intermolecular enzyme-substrate interactions. To address this problem we here apply a modified hybrid structure strategy for homology modeling of plant P450s. This allows for structural elucidation based on conserved motifs in the protein sequence and secondary structure predictions. We modeled the well-studied Sorghum bicolor cytochrome P450 CYP79A1 catalyzing the first step in the biosynthesis of the cyanogenic glucoside dhurrin. Docking experiments identified key regions of the active site involved in binding of the substrate and facilitating catalysis. Arginine 152 and threonine 534 were identified as key residues interacting with the substrate. The model was validated experimentally using site-directed mutagenesis. The new CYP79A1 model provides detailed insights into the mechanism of the initial steps in cyanogenic glycoside biosynthesis. The approach could guide functional characterization of other membrane-bound P450s and provide structural guidelines for elucidation of key structure-function relationships of other plant P450s.

Case study on human α1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate

Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme‐linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available ELISA kits. We observed that estimations of recombinant human ø1‐antitrypsin (rø1AT) titer by Coomassie‐stained SDS‐PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based on biolayer interferometry and reversed‐phase high‐performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off‐the‐shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers determined by ELISA kits cannot be trusted per se. Consequently, any ELISA kit to be used for determining recombinant protein titer must be validated by a different, preferably orthogonal method.

CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins

In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6% and ≈3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.

Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence

Methods for site-selective chemistry on proteins are in high demand for the synthesis of chemically modified biopharmaceuticals, as well as for applications in chemical biology, biosensors and more. Inadvertent N-terminal gluconoylation has been reported during expression of proteins with an N-terminal His tag. Here we report the development of this side-reaction into a general method for highly selective N-terminal acylation of proteins to introduce functional groups. We identify an optimized N-terminal sequence, GHHHn− for the reaction with gluconolactone and 4-methoxyphenyl esters as acylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acylation with the 4-methoxyphenyl esters. This Gly-Hisn tag adds the unique capability for highly selective N-terminal chemical acylation of expressed proteins. We anticipate that it can find wide application in chemical biology and for biopharmaceuticals.His-tagged proteins can undergo N-terminal acylation as an undesired side-reaction. Here, the authors utilize this to develop a method for highly selective acylation and further modification of peptides and proteins using an optimized His sequence and 4-methoxyphenyl esters as acyl donors.