miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c‐Met. We previously demonstrated that HGF α‐chain hairpin–loop, K1 domain and β‐chain are required for c‐Met signalling. The sequential phosphorylation of tyrosine residues, from c‐Met kinase domain to multidocking regions, is required for HGF‐signalling transduction. Herein, we provide evidence that the disconcerted activation of c‐Met tyrosine regions fails to induce biological functions. When human cells were incubated with ‘mouse HGF’, kinase domain activation (i.e. phospho‐Tyr‐1230/34/35) became evident, but the multidocking site (i.e. Tyr‐1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α‐chain, or of β‐chain, to human c‐Met was lower than that of human HGF, as evidenced by HGF–chimera assay. Notably, only four amino acid positions in HGF α‐chain hairpin–loop and K1 domain and six positions in β‐chain differed between human HGF and mouse HGF. The human‐specific amino acids (such as Gln‐95 in hairpin–loop, Arg‐134 in K1 domain and Cys‐561 in β‐chain) may be important for accurate c‐Met assembly and signalling transduction. Copyright © 2012 John Wiley & Sons, Ltd.     

Role of HGF/c‐Met in the treatment of colorectal cancer with liver metastasis

The system of hepatocyte growth factor (HGF) and its receptor c‐Met plays a critical role in tumor invasive growth and metastasis. The mortality rate of colorectal cancer (CRC), one of the most commonly diagnosed malignancies, is increased by it gradual development into metastasis, most frequently in the liver. Overexpression of c‐Met, the protein tyrosine kinase receptor for the HCF/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. In this study, we aimed to investigate the role of c‐Met in CRC liver metastasis and illustrate the clinical impact of regulating HGF/c‐Met signaling in patients with CRC liver metastasis. We found that (I) higher levels of c‐Met expression (mRNA and Protein) in CRC liver metastasis than primary CRC by assessing the patient tissue samples; (II) a positive correlation of c‐Met expression with tumor stages of CRC liver metastasis, as well as c‐Met expression in CRC, live metastasis concurred with regional lymph node metastasis; (III) the clinical impact of downregulation of HGF/c‐Met signaling on the reduction of proliferation and invasion in CRC liver metastasis. Therefore, we demonstrate that the regulation of HGF/c‐Met pathways may be a promising strategy in the treatment of patients with CRC liver metastasis.     

MicroRNA‐876‐5p inhibits cell proliferation,migration and invasion by targeting c‐Met in osteosarcoma

Recently, aberrant expression of miR‐876‐5p has been reported to participate in the progression of several human cancers. However, the expression and function of miR‐876‐5p in osteosarcoma (OS) are still unknown. Here, we found that the expression of miR‐876‐5p was significantly down‐regulated in OS tissues compared to para‐cancerous tissues. Clinical association analysis indicated that underexpression of miR‐876‐5p was positively correlated with advanced clinical stage and poor differentiation. More importantly, OS patients with low miR‐876‐5p level had a significant shorter overall survival compared to miR‐876‐5p high‐expressing patients. In addition, gain‐ and loss‐of‐function experiments demonstrated that miR‐876‐5p restoration suppressed whereas miR‐876‐5p knockdown promoted cell proliferation, migration and invasion in both U2OS and MG63 cells. In vivo studies revealed that miR‐876‐5p overexpression inhibited tumour growth of OS in mice. Mechanistically, miR‐876‐5p reduced c‐Met abundance in OS cells and inversely correlated c‐Met expression in OS tissues. Herein, c‐Met was recognized as a direct target of miR‐876‐5p using luciferase reporter assay. Notably, c‐Met restoration rescued miR‐876‐5p attenuated the proliferation, migration and invasion of OS cells. In conclusion, these findings indicate that miR‐876‐5p may be used as a potential therapeutic target and promising biomarker for the diagnosis and prognosis of OS.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Induction of insulin‐producing cells from umbilical cord blood‐derived stromal cells by activation of the c‐Met/HGF axis

Efficient and effective therapies are required for diabetes mellitus. The use of adult stem cells for treating diabetes represents a major focus of current research. We have attempted to differentiate adult stem cells produced from umbilical cord blood‐derived stromal cells into insulin‐producing cells (IPCs). By activating the c‐Met/HGF axis through temporal hypoxia treatment and hepatocyte growth factor (HGF) supplementation, our protocol resulted in the differentiation of cells into functional pancreatic endocrine cells with increased viability. Glucose stimulation test results showed that significantly greater amounts of C‐peptide and insulin were released from the differentiated cells than from undifferentiated cells. These IPCs were capable of reversing the hyperglycemia of diabetic mice. In conclusion, targeting the c‐Met/HGF axis can be considered an effective and efficient means of obtaining IPCs from adult stem cells.     

ADAM10 correlates with uveal melanoma metastasis and promotes in vitro invasion

Uveal melanoma (UM) is a rare ocular tumor that may lead to deadly metastases in 50% of patients. A disintegrin and metalloproteinase (ADAM)10, ADAM17, and the HGF‐receptor c‐Met support invasiveness in different tumors. Here, we report that high ADAM10, MET, and, to a lesser extent, ADAM17 gene expression correlates with poor progression‐free survival in UM patients (hazard ratio 2.7, 2.6, and 1.9, respectively). About 60% of primary UM expresses c‐Met and/or ADAM10 proteins. Four UM cell lines display high levels of ADAM10 and ADAM17, which constitutively cleave c‐Met, inducing the release of soluble c‐Met. ADAM10/17 pharmacological inhibition or gene silencing reduces c‐Met shedding, but has limited impact on surface c‐Met, which is overexpressed. Importantly, ADAM10 silencing inhibits UM cell invasion driven by FCS or HGF, while ADAM17 silencing has a limited effect. Altogether our data indicate that ADAM10 has a pro‐invasive role and may contribute to UM progression.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

CLIP‐170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus‐end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward‐directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP‐170, as well as the association of CLIP‐170 to MT plus‐ends. In response to HGF, entry of Met into Rab4‐positive endosomes results in Golgi‐localized γ‐ear‐containing Arf‐binding protein 3 (GGA3) and CLIP‐170 recruitment to an activated Met RTK complex. We conclude that CLIP‐170 co‐ordinates the recycling and the transport of Met‐positive endocytic vesicles to plus‐ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration.      

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

N‐linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N‐linked glycosylation patterns in a CHO‐S cell fed‐batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High‐throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N‐linked glycosylation patterns during a fed‐batch process as a function of time as well as the manipulated variables. A constant and varying mAb N‐linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model‐based evaluation of feeding regimes using high‐throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135–1148, 2016     

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody,AMG102

The hepatocyte growth factor (HGF)/Met signalling pathway is up‐regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti‐HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U‐87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.     

Src and FAK mediate cell–matrix adhesion‐dependent activation of met during transformation of breast epithelial cells

Cell–matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand‐independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non‐neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto‐activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with β1 integrins and is co‐localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src‐dependent phosphorylation of Met requires cell–matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src‐induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF‐dependent and Src/integrin‐dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168–1181, 2009. © 2009 Wiley‐Liss, Inc.     

N‐linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis

Vacuolar sorting receptors (VSRs) in Arabidopsis mediate the sorting of soluble proteins to vacuoles in the secretory pathway. The VSRs are post‐translationally modified by the attachment of N‐glycans, but the functional significance of such a modification remains unknown. Here we have studied the role(s) of glycosylation in the stability, trafficking and vacuolar protein transport of AtVSR1 in Arabidopsis protoplasts. AtVSR1 harbors three complex‐type N‐glycans, which are located in the N‐terminal ‘PA domain’, the central region and the C‐terminal epidermal growth factor repeat domain, respectively. We have demonstrated that: (i) the N‐glycans do not affect the targeting of AtVSR1 to pre‐vacuolar compartments (PVCs) and its vacuolar degradation; and (ii) N‐glycosylation alters the binding affinity of AtVSR1 to cargo proteins and affects the transport of cargo into the vacuole. Hence, N‐glycosylation of AtVSR1 plays a critical role in its function as a VSR in plants.     

Met degradation by SAIT301, a Met monoclonal antibody,reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression

Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy.     

Allosteric peptide activators of pro-hepatocyte growth factor stimulate Met signaling

Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/β-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like β-chain (HGF β), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the β-chain stimulate Met phosphorylation by pro-HGF to levels that are ～25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF β binding to Met, resulting in a K(D)(app) of 1.6 μm, only 8-fold weaker than the Met/HGF β-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like β-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.