Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug.

Recurrent hemichorea in an adolescent with systemic lupus erythematosus and previous ipsilateral cerebral infarction.

Systemic lupus erythematosus (SLE) can present with movement disorders, among which chorea is closely associated with antiphospholipid (aPL) antibodies. Brain imaging results obtained in patients with chorea are generally inconsistent with the clinical manifestation of chorea; moreover, medical tests for hemichorea, which are expected to reveal distinct localization, may show negative findings. Herein, we present a case of a 15-year-old girl with SLE who had a history of left cerebral infarction; tests revealed elevated aPL levels, and she developed recurrent left hemichorea 2 years later.

Cell activation-based screening of natively paired human T cell receptor repertoires.

Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR.

Quality Control: Chain Pairing Precision and Monitoring of Cross-Sample Contamination: A Method by the AIRR Community.

New approaches in high-throughput analysis of immune receptor repertoires are enabling major advances in immunology and for the discovery of precision immunotherapeutics. Commensurate with growth of the field, there has been an increased need for the establishment of techniques for quality control of immune receptor data. Our laboratory has standardized the use of multiple quality control techniques in immunoglobulin (IG) and T-cell receptor (TR) sequencing experiments to ensure quality control throughout diverse experimental conditions.

Immortalization and functional screening of natively paired human T cell receptor repertoires.

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant.

The impact of sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases.

Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme.

Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production.

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of β-1,4- and β-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation.

Antibody glycoengineering strategies in mammalian cells.

As a key parameter impacting functional and structural heterogeneity, protein glycosylation is a critical quality attribute for antibody biotherapeutic manufacturing. The glycan patterns on recombinant antibodies, particularly on the conserved fragment crystallizable (Fc) region, can have significant effects on an antibody's functional activities including clearance rate, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activity. In this review, we examined specific glycan attachments (fucosylation, sialylation, galactosylation, high-mannose, and bisecting glycans) and their importance to antibody properties.

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation.

Proline-Rich Chaperones Are Compared Computationally and Experimentally for Their Abilities to Facilitate Recombinant Butyrylcholinesterase Tetramerization in CHO Cells.

Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents. Efficient assembly of human BChE into tetramers requires an association with proline-rich peptide chaperones. In this study, the incorporation of different proline-rich peptide chaperones into BChE is investigated computationally and experimentally.

SnapShot: N-Glycosylation Processing Pathways across Kingdoms.

Post-translational modification of proteins with carbohydrates shapes their localization and function. This SnapShot presents the core pathways from different organisms that install these complex and highly variable structures.

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities.

Glycoengineering of CHO Cells to Improve Product Quality.

Chinese hamster ovary (CHO) cells represent the predominant platform in biopharmaceutical industry for the production of recombinant biotherapeutic proteins, especially glycoproteins. These glycoproteins include oligosaccharide or glycan attachments that represent one of the principal components dictating product quality. Especially important are the N-glycan attachments present on many recombinant glycoproteins of commercial interest.

A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well.

Integrated Genome and Protein Editing Swaps α-2,6 Sialylation for α-2,3 Sialic Acid on Recombinant Antibodies from CHO.

Immunoglobin G with α-2,6 sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. However, production of antibodies with α-2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N-glycan site and the presence of exclusively α-2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α-2,6 sialyltransferase produced IgG with significant levels of both α-2,6 and α-2,3 sialylation.

Transparent deoxyribonucleic acid substrate with high mechanical strength for flexible and biocompatible organic resistive memory devices.

Biocompatible deoxyribonucleic acid (DNA), with high mechanical strength, was employed as the substrate for a Ag nanowire (Ag NW) pattern and then used to fabricate flexible resistor-type memory devices. The memory exhibited typical write-once-read-many (WORM)-type memory features with a high ON/OFF ratio (10), long-term retention ability (10 s) and excellent mechanical endurance.

Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation.

Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO increased by 26%.

Assessment of the coordinated role of ST3GAL3, ST3GAL4 and ST3GAL6 on the α2,3 sialylation linkage of mammalian glycoproteins.

In this research, we examined which genes are involved in N-linked sialylation in Chinese Hamster Ovary (CHO) cells using siRNA knockdown approaches. Three genes from the sialyltransferase family (ST3GAL3, ST3GAL4 and ST3GAL6) were chosen as knockdown targets with siRNA applied to reduce their expression. Single, double and triple gene knockdowns were investigated, and the reduction levels of sialylation on the total cell lysate were monitored by enzyme-linked lectin absorption assays (ELLA) and sialic acid quantification with high performance liquid chromatography (HPLC).

CHO microRNA engineering is growing up: recent successes and future challenges.

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies.

Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability.

In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane.

Primary ovary transitional cell carcinoma after renal transplantation.

Transitional cell carcinoma (TCC) of the urinary tract is the most frequent malignancy following renal transplantation reported in Taiwan. A 67-year-old female underwent bilateral nephrouretectomy and bladder cuff excision because of bilateral hydronephrosis 5 years after cadaveric renal transplantation. The pathologic report was only atrophied kidney.

Baculovirus as an avian influenza vaccine vector: differential immune responses elicited by different vector forms.

Baculovirus is an enveloped virus that infects insects in nature and has emerged as a novel vaccine vector. We previously constructed a recombinant baculovirus displaying the hemagglutinin protein (HA) of avian influenza virus (AIV) on the viral envelope (Bac-HA64), and demonstrated the induction of humoral responses in immunized mice. To improve the vector design and explore how the vector forms influence the vaccine efficacy, we constructed two more baculoviruses Bac-CHA and Bac-CHA/HA64.

Enterovirus 71 virus-like particle vaccine: improved production conditions for enhanced yield.

To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells.