Anders Holmgaard Hansen

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.

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks underlying glycosylation and the vast number of different glycans that can be synthesized in a host cell. Computational modeling offers an intriguing option to rationally guide glycoengineering, but the high parametric demands of current modeling approaches pose challenges to their application. Here we present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles on EPO, IgG as well as the endogenous secretome following glycosyltransferase knock-out in different Chinese hamster ovary (CHO) cell lines. Our approach offers a flexible and user-friendly platform that can serve as a basis for powerful computational engineering efforts in mammalian cell factories for biopharmaceutical production.

A new high-throughput LC-MS method for the analysis of complex fructan mixtures

In this paper, a new liquid chromatography-mass spectrometry (LC-MS) method for the analysis of complex fructan mixtures is presented. In this method, columns with a trifunctional C18 alkyl stationary phase (T3) were used and their performance compared with that of a porous graphitized carbon (PGC) column. The separation of fructan isomers with the T3 phase improved clearly in comparison with the PGC phase, and retention times were lower and more stable. When the T3-based method was applied on a wheat grain extract, multiple fructan isomers could be discerned, even for fructans with a degree of polymerization of 10. This indicates that wheat grain fructans do not, or not only, have a simple linear structure. The presented method paves the way for elucidation of fructan structures in complex mixtures that contain many structural isomers.

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.

Building a fructan LC–MS2 library and its application to reveal the fine structure of cereal grain fructans

A liquid chromatography-mass spectrometry (LC-MS) library is presented containing the relative retention times of 28 fructan oligomers and MS2 spectra of 18 of them. It includes the main representatives of all fructan classes occurring in nature and with a degree of polymerization between three and five. This library enables a rapid and unambiguous detection of these 18 fructan structures in any type of sample without the need for fructan purification or the synthesis of fructan standards. Its wide applicability is demonstrated by the analysis of fructans in a set of cereal flour samples. Marked differences were observed in the types of fructans present in oat, barley, rye, spelt and wheat flour. A putative link between the accumulation of certain fructan types and cereal phylogeny is described.

Liquid chromatography/mass spectrometry analysis of branched fructans produced in vitro with 13C‐labeled substrates

RATIONALE Fructans are carbohydrates predominantly based on fructose which are generally considered to be soluble dietary fibers with health-promoting properties. It is known that the nutritional properties of fructans are affected by their structure. This study focused on structural determination of branched fructans, as the most important dietary fructans are branched graminan-type fructans. METHODS Branched fructans were synthesized enzymatically by incubation of a heterologously expressed sucrose:fructan 6-fructosyltransferase (6-SFT) from Pachysandra terminalis with native or (13)C-labeled substrates. Liquid chromatography/mass spectrometry (LC/MS) was used for the structural identification of branched fructans. The MS(2) fragmentation of these compounds is described for the first time. Analytes were charged by electrospray ionization in negative mode and a quadrupole mass analyzer was used for MS(2) analysis. RESULTS The MS(2) fragmentation patterns of branched and linear fructans were shown to differ and distinctive ion formation allowed differentiation between all branched fructan isomers formed. P. terminalis 6-SFT preferred extending the existing fructan branch rather than creating a new branch. CONCLUSIONS The MS(2) fragmentation patterns described in the current paper now allow rapid screening of large sample sets for the presence of branched, graminan-type fructans. Furthermore, the data enables the characterization of fructan-metabolizing enzymes by identification of the fructan structures produced by in vitro reactions as described here for P. terminalis 6-SFT.

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.

De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions

Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading to the formation of high-value compounds. In the present study, we systematically maximize the heterologous expression of six different plant-derived CYP genes in Escherichia coli, using a workflow based on C-terminal fusions to the green fluorescent protein. The six genes can be over-expressed in both K- and B-type E. coli strains using standard growth media. Furthermore, sequences encoding a small synthetic peptide and a small bacterial membrane anchor markedly enhance the expression of all six genes. For one of the CYPs, the length of the linker region between the predicted N-terminal transmembrane segment and the soluble domain is modified, in order to verify the importance of this region for enzymatic activity. The work describes how membrane bound CYPs are optimally produced in E. coli and thus adds this plant multi-membered key enzyme family to the toolbox for bacterial cell factory design.

A novel low-parameter computational model to aid in-silico glycoengineering

Background Glycosylation is a key post-translational modification that can affect critical properties of proteins produced in biopharmaceutical manufacturing, such as stability, therapeutic efficacy or immunogenicity. However, unlike a protein’s amino acid sequence, glycosylation is hard to engineer since it does not follow any direct equivalent of a genetic code. Instead, its complex biogenesis in the Golgi apparatus (Figure 1A) integrates a variety of influencing factors most of which are only incompletely understood. Various attempts have been undertaken so far to computationally model the process of glycosylation, but due to the high parametric demand of most of these models, it has been challenging to leverage these models for glycoengineering purposes. Consequently, industrial glycoengineering is still largely carried out using costly and time-consuming trial-and-error strategies and could greatly benefit from computational models that would better meet the requirements for industrial utilization. Here, we introduce a novel approach combining constraints-based and stochastic techniques to derive a computational model that can predict the effects of gene knockouts on protein glycoprofiles while requiring only minimal a-priori parameter input.