42 results about "Cricetulus" patented technology

The present disclosure provides a Chinese Hamster Ovary (CHO) cell comprising one or more exogenous nucleotide sequences, wherein the one or more exogenous nucleotide sequences is inserted into a chromosomal locus of the CHO cell, wherein the chromosomal locus is located within contig NW_023276805.1 of chromosome X. Also provided herein are methods of producing a CHO cell capable of expressing a sequence of interest, methods of expressing a sequence of interest comprising culturing the CHO cell as described herein, and kits comprising the CHO cell as described herein.

Provided are pharmaceutical formulations comprising a recombinant acid α-glucosidase, wherein the recombinant acid α-glucosidase is expressed in Chinese hamster ovary (CHO) cells and comprises an increased content of N-glycan units bearing one or two mannose-6-phosphate residues when compared to a content of N-glycan units bearing one or two mannose-6-phosphate residues of alglucosidase alfa; at least one buffer selected from the group consisting of a citrate, a phosphate and combinations thereof; and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, wherein the formulation has a pH of from about 5.0 to about 7.0. Also provided are methods of treating Pompe disease using these pharmaceutical formulations.

The present invention is related to a method of determining suitability of at least one Chinese Hamster Ovary (CHO) test cell line for optimal heterologous protein production, the method comprising:(a) determining a test methylation profile from genomic material obtained from the CHO test cell line; and(b) comparing the test methylation profile obtained from (a) with a reference methylation profile, wherein the reference methylation profile comprises the methylation status of more than one CpG site from at least one CHO reference cell line that displays at least one phenotype of interest for optimal heterologous protein production,wherein a significant similarity in the test methylation profile of (a) compared to the reference methylation profile, is indicative of the CHO test cell line being suitable for optimal heterologous protein production and wherein the test methylation profile and reference methylation profile are from CpG sites from the CHO cell genome and are determined using DNA methylation-bead-based array.

The present disclosure relates to methods, cells, and compositions for producing a product of interest, e.g., a recombinant protein. In particular, the present disclosure provides improved mammalian cells expressing the product of interest, where the cells (e.g., Chinese Hamster Ovary (CHO) cells) have reduced or eliminated activity, e.g., expression, of certain host cell proteins, e.g., enzymes including, but not limited to, certain lipases, esterases, and / or hydrolases.

One approach to the exploration method includes calculating a TAD score representing transcriptional activity for each TAD present in the genome and selecting TADs based on the TAD scores. Another approach includes analyzing a genome obtained from cells expressing a foreign gene and selecting the boundaries of regions containing the foreign gene. Cell preparation involves inserting the target gene into the regions identified by the exploration method. Cell sorting involves calculating a TAD score representing transcriptional activity for each TAD present in the genome, selecting TADs based on the TAD scores, and selecting cells containing the target gene in the selected TADs. The cells are derived from Chinese hamsters and are cells in which the target gene has been inserted into at least one specified region. The method for manufacturing the cell product includes culturing the cells to express the target gene.

The invention relates to a method for improving PD-1 titer produced by Chinese hamster ovary cells. The method comprises the following steps: inoculating the Chinese hamster ovary cells into an initial culture medium for culturing; after the culture is performed for 48 hours, feeding a fed-batch culture medium every day, and maintaining the glucose concentration in the culture medium to be 1.3-1.7 g / L, the galactose concentration to be 0.6-1.0 g / L and the L-sodium lactate concentration to be 14-16 mM; the culture temperature is reduced, a fed-batch culture medium is fed every day, the glucose concentration in the culture medium is maintained at (2-2.8) g / L, the galactose concentration is maintained at (1.7-2.2) g / L, and cells are harvested. Galactose is introduced to serve as a second carbon source, and control over the culture initial metabolism environment and the carbon source in the culture stage is combined, so that the maximum accumulation amount of lactic acid is obviously reduced, the cell culture period is remarkably prolonged, and the maximum titer of the PD-1 antibody is remarkably improved compared with an existing process.

The invention provides an expression vector, an expression system and a preparation method of a recombinant larosidase protein, and belongs to the technical field of recombinant protein preparation, the recombinant expression vector of the larosidase protein provided by the invention comprises an initial vector and a codon-optimized human-derived larosidase coding gene; the nucleotide sequence of the human-derived larosidase coding gene is as shown in SEQ ID NO. 1; according to the recombinant expression system, Chinese hamster ovary cells are taken as host cells, and the recombinant expression vector is transfected. According to the invention, a gene sequence more suitable for expression of Chinese hamster ovary cells is obtained through codon optimization, and then transposon-mediated stable integration is carried out; high-density suspension culture and downstream purification processes are utilized to realize efficient and stable expression of the recombinant larosidase in CHO cells, and the recombinant larosidase with high yield, high activity and high purity can be obtained.

The present invention relates to an in vitro method for predicting the chronological age of a test Chinese Hamster Ovary (CHO) cell or population of test CHO cells, the method comprising the steps of: (a) determining the test methylation level of a set of specific CpG sites from genomic material obtained from the test CHO cell or from the population of test CHO cells from a CHO cell line using a DNA-methylation bead-based array; (b) comparing the test methylation level determined from step (a) with a reference methylation level of the same set of specific CpG sites from an age-correlated reference sample, thereby establishing the epigenetic age and predicting the chronological age of the test CHO cell or the population of test CHO cells wherein the set of specific CpG sites comprises at least 10%, preferably 30%, more preferably 50% of the CpG sites indicated in Table 1.

The invention provides a cell genome site capable of being used for targeted integration of exogenous nucleic acid molecules. The site is located in a nucleic acid sequence corresponding to a cell genome NC048595.1: 454997734-455024715 of a Chinese hamster ovary CHO-K1. The cell genome sites can integrate foreign protein genes and perform stable expression. The invention also provides stable and high-yield targeted integrated cells, such as host cells derived from Chinese Hamster Ovary (CHO), methods for their production and uses thereof.

The invention provides an expression vector of a mild hypothermia response type hybrid promoter, a host cell and application of the expression vector and the host cell, and belongs to the technical field of genetic engineering and biological pharmacy. The hybrid promoter comprises a low-temperature response enhancer element of a Chinese hamster heat shock protein 90 (HSP90) gene and a core promoter element of a cytomegalovirus (CMV) immediate early gene. The promoter has low activity at a standard culture temperature (37 DEG C), is specifically and efficiently activated at a mild hypothermia (30-33 DEG C), and can significantly improve the expression yield of recombinant protein under a mild hypothermia culture condition.

The invention discloses a multiple PCR-capillary electrophoresis detection system for identifying 13 species and a special primer combination thereof. The primer combination is composed of 26 kinds of DNA (deoxyribonucleic acid) molecules as shown in SEQ ID No. 1 to SEQ ID No. 26. The primer combination is adopted to construct a multiple PCR-capillary electrophoresis detection system, DNA (such as DNA of blood or meat) of 13 species such as chicken, duck, sheep, pig, cattle, mouse, rat, cat, dog, donkey, horse, human and Chinese hamster can be simultaneously identified in a single-tube reaction, the sensitivity to different target DNA is 0.001-0.05 ng, and compared with other researches, the primer combination has obvious advantages in sensitivity and species identification quantity. The method is low in cost and high in efficiency, and has important application value.

The present disclosure relates to a perfusion-evolved Chinese hamster ovary cell line and a method of generating said cell line.

The application discloses application of a fatty acid in improving concentration of a therapeutic antibody expressed by CHO cells. The application relates to the technical field of protein engineering, and the exact composition of the additive is eicosanoid C20:2. In the culture process of Chinese hamster ovary (CHO) cells for expressing a therapeutic antibody, the addition of C20:2 can improve the stress resistance of the CHO cells in the culture process, thereby effectively improving the yield of the therapeutic antibody, and the quality of the antibody is not affected.

Type-C endogenous retroviruses (ERVs) embedded in Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and / or retroviral-like particles in the culture supernatant. Although evidence for the infectivity of these particles is missing. their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding viral RNA genomes that were loaded into retroviral-like particles. Also, sequence analysis of the genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption / alteration of the gag gene of the expressed ERV group using CRISPR-Cas9 genome editing. Comparison of CRISPR-derived mutations at the DNA and mRNA level led to the identification of a single ERV locus responsible for the release of viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function mutation in this particular ERV locus showed a reduction of viral RNA-containing particles in the cell supernatant by over 250-fold. Notably, ERV mutagenesis did not compromise cell growth, cell size or recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during biopharmaceutical manufacturing.

The invention relates to a method of predicting quality of a heterologous protein to be produced from a test Chinese Hamster Ovary (CHO) cell, the method comprising the steps of: (a) determining a test methylation profile of one or more pre-selected methylation CpG sites within the DNA of the test CHO cell; (b) comparing the test methylation profile obtained from (a) with a reference methylation profile, wherein the reference methylation profile comprises the methylation status of the pre-selected methylation CpG sites from at least one CHO reference cell line that is capable of producing a known, preferably good, quality heterologous protein; wherein a significant similarity in the test methylation profile of (a) compared to the reference methylation profile, is indicative of the heterologous protein produced by the test cell being of known, preferably good, quality; and wherein a significant difference in the test methylation profile of (a) compared to the reference methylation profile, is indicative of the heterologous protein produced by the test cell being of unknown or poor quality; wherein the test and reference methylation profiles are determined using bead-based DNA methylation array; and wherein the quality of the heterologous protein is based on the presence of at least one protein quality metric; and the pre-selected methylation CpG sites are selected from Tables 4 and 5.

The present invention relates to a mammalian non-human cell line, specifically Chinese hamster ovary (CHO) cells, that is genetically modified to express poxvirus host range genes CP77, K1L and / or SPI-1 which are not expressed in MVA, and to the use of said cell line in the reproduction of MVA.

The invention provides a domestication culture medium composition suitable for Chinese hamster ovary cells CHO-K1, a domestication method and application of the domestication culture medium composition. Wherein the composition for domesticating the CHO-K1 comprises a yeast hydrolysate and L-glutamine; wherein the content of the yeast hydrolysate is 0.6 to 2wt%, and the content of the L-glutamine is 0.04 to 0.25 wt%. The composition provided by the invention can realize rapid and efficient CHO-K1 suspension domestication through the yeast hydrolysate and L-glutamine with specific composition, and the domesticated cells are high in viability, high in proliferation speed, single in dispersion, uniform in size, round and transparent.

The invention discloses recombinant collagenase as well as a preparation method and application thereof, and belongs to the technical field of biochemical engineering. The recombinant collagenase provided by the invention is prepared from HmCol and / or CbCol, the amino acid sequence of the HmCol is as shown in SEQ ID NO: 1; the amino acid sequence of the CbCol is as shown in SEQ ID NO: 2. Recombinant collagenase hydrolytic activity experiment results show that the expressed recombinant collagenase HmCol and CbCol can recognize recombinant collagen of different sources, including fragments of human III-type collagen recombined and expressed by escherichia coli, yeast and Chinese hamster ovary cells (CHO), so that the specificity and efficient hydrolysis of the collagen are realized, and the recombinant collagenase HmCol and CbCol can be used for identifying the recombinant collagen of different sources. The material has good application prospects in the aspects of scar tissue repair, low-temperature cold compress debridement and the like, and can be used as a raw material for preparing skin repair products.

The invention provides bidirectional expression vectors comprising Chinese hamster ovary elongation factor 1-α (CHEF1) transcriptional regulatory DNA elements, a gene of interest (GOI), a minimal cytomegalovirus (minCMV) and a selectable marker (SM) and / or a human adenovirus tripartite leader (AdTPL) sequence. The invention also provides method for increasing heterologous protein expression in a host cell comprising culturing the host cell the bidirectional expression vector(s).

The present disclosure relates to methods, cells and compositions for producing a product of interest, e.g., a recombinant protein. In particular, the present disclosure provides improved mammalian cells expressing the product of interests, where the cells (e.g., Chinese Hamster Ovary (CHO) cells) have modulated lactogenic activity. The present disclosure also relates to methods and compositions for modulating pyruvate kinase muscle (PKM) expression (e.g., PKM-1 expression) in a mammalian cell to thereby reduce or eliminate the lactogenic activity of the cell, as well compositions comprising a cell having reduced or eliminated lactogenic activity and methods of using the same.

This invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the invention includes compositions and methods for improved expression of antibodies including bispecific antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing an expression-enhancing locus.

Provided is a promoter having high foreign gene expression-enhancing activity in host cells such as cultured mammalian cells. An approach for using the promoter to enhance the production of a foreign protein to be used as a protein-based pharmaceutical product is the provision of a polynucleotide that is a Chinese hamster-derived gene promoter, the polynucleotide comprising a nucleotide sequence that is selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, or has 85% or higher sequence identity to each of the sequences, or a partial sequence of the nucleotide sequence.

The invention belongs to the technical field of gene engineering, and particularly relates to application of Hoxa10 gene in improving expression of exogenous recombinant protein of mammalian cells. The nucleotide sequence of the Hoxa10 gene is shown as SEQ ID No.1. The invention discloses for the first time that the Hoxa10 gene can be used for improving the expression of exogenous recombinant proteins of mammalian cells: the Hoxa10 is used as a gene engineering modification target of recombinant protein expression, a recombinant vector pWTY-Hoxa10 for overexpressing the Hoxa10 is constructed, then the recombinant vector for overexpressing the Hoxa10 is transfected to Chinese hamster ovary (CHO) cells, and the expression of the exogenous recombinant proteins of the mammalian cells is improved. A recombinant CHO cell line for stable overexpression of Hoxa10 is obtained, and the growth performance of host cells is improved after overexpression of Hoxa10. When the recombinant cell is used for recombinant protein expression, the expression quantity of the recombinant protein is obviously improved.

To provide compositions and methods for making antibodies based on the use of expression-enhancing loci.SOLUTION: This invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the invention includes compositions and methods for improved expression of antigen-binding proteins including monospecific and bispecific antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing multiple expression-enhancing loci.SELECTED DRAWING: None

This invention relates to a method for constructing a biomarker for a first target phenotype of CHO cells, the method comprising the steps of: (a) determining the methylation values ​​of all CpG sites in the genomic DNA of a CHO cell population; the CHO cell population representing the first target phenotype and being part of a training sample; (b) identifying a set of specific CpG sites from the training sample of step (a), the CpG sites having reliable methylation values; and (c) applying a penalized regression model to perform penalized regression, using the methylation values ​​obtained in step (a) as input and the target phenotype associated with the training sample as the dependent variable; thereby obtaining specific CpG sites with corresponding weight coefficients and the intercept of the linear model equation, which are used as parameters to define the biomarker of the first target phenotype.

Disclosed herein are expression cassettes, expression vectors, dual vector systems, and mammalian host cells, including but not limited to Chinese hamster ovary (CHO) cells, comprising the foregoing, for the expression of certain antibody modalities, wherein each expression vector employs a promoter set optimized for the antibody modality. Also disclosed herein are methods of producing certain antibody modalities using these mammalian host cells.

The present invention relates to novel affinity ligands that bind to human tissue plasminogen activator (tPA). The novel affinity ligands of the present invention are powerful tools because they allow efficient purification methods for human tPA via affinity chromatography. In particular, the affinity ligands are powerful in the purification of recombinant human tPA that was produced in Chinese hamster ovary cells because the affinity ligands of the invention do not detectably bind to hamster tPA. Accordingly, the present invention also relates to a novel process of isolating and purifying tPA, specifically tPA from CHO cells.

The present invention relates to glycosylated FC variants of which binding affinity for human FcγRs is removed, to minimize off-target toxicity of an antibody against an antigen. Novel human antibody Fc domain variants of the present invention, which were discovered using Chinese hamster ovary (CHO) cells having a very similar sugar profile to humans, have significantly reduced binding to Fc gamma receptors, compared to wild-type human antibody Fc domain and conventional S228P or S228P / L235E variants, and are variants of which pH-dependent binding affinity for FcRn and thermal stability are maintained and binding affinity for all FcγRs is completely removed. Therefore, the variants can be used to reduce the toxicity and enhance the efficacy of therapeutic protein drugs and to maintain the half-life of diagnostic / research substances and remove target toxicity.

This invention relates to site-specific integration and expression of recombinant proteins in eukaryotic cells. In particular, the invention includes compositions and methods for improved expression of antigen-binding proteins including monospecific and bispecific antibodies in eukaryotic cells, particularly Chinese hamster (Cricetulus griseus) cell lines, by employing multiple expression-enhancing locus.

The present invention provides a method for determining the suitability of at least one Chinese hamster ovary (CHO) test cell line for optimal heterologous protein production, the method comprising: (a) determining a test methylation profile from genomic material obtained from a CHO test cell line; (b) comparing the test methylation profile obtained from (a) with a reference methylation profile, the reference methylation profile comprising the methylation status of two or more CpG sites from at least one CHO reference cell line exhibiting at least one phenotype of interest for optimal heterologous protein production; Including, wherein a significant similarity of the test methylation profile of (a) compared to the reference methylation profile indicates that the CHO test cell line is suitable for optimal heterologous protein production, and wherein the test methylation profile and the reference methylation profile are from CpG sites from the CHO cell genome and are determined using a DNA methylation bead-based array.