58 results about "Chinese hamster" 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.

Disclosed herein are expression systems for producing multi-specific antibodies having only one Fab or having two Fabs that are identical, host cells comprising such expression systems, including but not limited to Chinese hamster ovary (CHO) cells, and methods for producing multi- specific antibodies utilizing such expression systems.

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 is applicable to the technical field of biology, provides a CHO (Chinese hamster ovary) cell culture method combining continuous feeding and fed-batch culture, and aims to improve the cell density and the product yield. The method comprises two stages: a continuous feeding proliferation stage: at the initial stage of cell culture, adopting a continuous feeding mode to increase the cell density to a first target density; and a dynamic batch feeding stage: when the cell density reaches the first target density, converting into a dynamic batch feeding mode based on real-time monitoring parameters, and increasing the cell density to be higher than second target density through a dynamic feeding strategy. Compared with a traditional process, the cell density and the protein yield are improved, accumulation of inhibitory metabolic byproducts such as lactic acid is effectively reduced through a dynamic feeding strategy, the cell culture environment is improved, and the method is suitable for production of high-value biological products such as monoclonal antibodies and vaccines and has a wide market prospect.

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 relates to a transfection reagent for CHO (Chinese hamster ovary) cell DNA (deoxyribonucleic acid) transfection, and the formula of the transfection reagent comprises N-(2, 3-dioleoyloxy-1-propyl) trimethylamine methyl sulfate, polyethyleneimine, N-methyl pyrrolidone and pure water. The invention also relates to a preparation method of the transfection reagent, which comprises the following steps: S1, mixing 150-250g / L of N-methyl pyrrolidone and pure water to 1L to obtain a solution; and S2, when the temperature of the solution is reduced to 30 DEG C or below, sequentially adding 800 to 1200 mg / L of N-(2, 3-dioleoyloxy-1-propyl) trimethylamine methyl sulfate and 400 to 600 mg / L of polyethyleneimine, and uniformly mixing, so as to obtain the transfection reagent. According to the invention, the permeability of transfected DNA to cell membranes is improved, so that the efficiency of PEI and DNA compounds entering cells is improved; meanwhile, according to the scheme, the dosage of the PEI is relatively reduced, on one hand, the toxicity of the PEI to cells is reduced, and on the other hand, the compound of the PEI and the transfection DNA can efficiently release the transfection DNA in the cells, so that the overall transfection efficiency is improved.

The present invention relates to the field of biomedicine and discloses a method for preparing a formaldehyde-crosslinked recombinant herpes zoster vaccine, comprising the following steps: S1. expressing varicella-zoster virus glycoprotein E in Chinese hamster ovary cells using DNA recombination technology; S2. diluting the herpes zoster recombinant protein with a low-temperature buffer to a concentration of less than 1.0 mg / ml; S3. mixing the formaldehyde solution with the low-temperature buffer to obtain a crosslinking solution with a formaldehyde concentration of 0.4% to 2%; S4. mixing the diluted herpes zoster recombinant protein solution with the crosslinking solution for a crosslinking reaction to ensure sufficient reaction between the protein and the formaldehyde crosslinker; S5. terminating the crosslinking reaction by exchanging the solution with PBS buffer. Through formaldehyde crosslinking technology, low-temperature mixing, and precise control of the crosslinking reaction, the invention improves the immunogenicity and stability of the vaccine, effectively enhancing the vaccine's effectiveness during long-term storage and transportation.

The invention relates to a method of culturing mammalian cells expressing a heterologous protein in a perfusion cell culture comprising increasing the potassium concentration and decreasing the molar ratio of sodium to potassium to reduce wasteful cell bleed and to increase protein production. The invention further relates to a serum-free perfusion medium comprising a high potassium ion concentration and a low molar ratio of sodium to potassium and to the use of this medium for use in culturing cells in a perfusion culture during production phase or for reducing the cell bleed volume during production phase.

Purified recombinant polypeptides isolated from Chinese hamster ovary host cells, including antibodies, such as therapeutic antibodies, and methods of making and using such polypeptides are provided.

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 invention relates to the technical field of transgenosis, in particular to a method for knockout of a Prkdc gene based on a CRISPR / Cas9 technology to construct an immunodeficient Chinese hamster. According to the invention, the CDS region of the Chinese hamster Prkdc gene is knocked out by utilizing a CRISPR / Cas9 technology, gRNA for targeted knockout of the Chinese hamster Prkdc gene is provided, and the application of a gene editing technology in the Chinese hamster is widened. The method for constructing the Prkdc gene knockout Chinese hamster animal model by using the CRISPR / Cas9 system has the advantages of high accuracy, simplicity, feasibility and short period, and provides service for research on immunodeficiency animal models and pathogenesis of human tumor diseases. The invention detects that the expression of T and B lymphocytes is reduced, which indicates that the Chinese hamster immunodeficiency animal model is successfully prepared. The application range of the Chinese hamster is widened, and a new species is provided for an immunodeficiency animal model.

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.

A method for producing biopharmaceuticals such as human antibodies. A Chinese hamster artificial chromosome that enables introduction of a foreign DNA of a target protein or the like in a site-directed manner into one or a plurality of DNA sequence insertion sites; a Chinese hamster artificial chromosome vector with foreign DNA of a target protein or the like introduced into the Chinese hamster artificial chromosome; and a mammalian cell with the Chinese hamster artificial chromosome vector introduced.

Disclosed herein are expression systems for producing multi-specific antibodies having only one Fab or having two Fabs that are identical, host cells comprising such expression systems, including but not limited to Chinese hamster ovary (CHO) cells, and methods for producing multi- specific antibodies utilizing such expression systems.

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 discloses an SSR4 gene edited CHO (Chinese Hamster Ovary) cell line as well as a preparation method and application thereof. The preservation number of the cell line is CCTCC (China Center For Type Culture Collection) NO: C2025176. An endogenous strong promoter (CMV) and a resistance selection marker are carried by a lentiviral vector, stable high expression (greater than 5 times) of SSR4 in CHO-S cells is realized, the cell growth rate is not influenced, the processing efficiency of glycoprotein in endoplasmic reticulum is further optimized, the proportion of HM glycotypes is remarkably reduced, excessive intervention of a traditional method on end modification of a carbohydrate chain is avoided, and the method has a good application prospect. Finally, accurate control of antibody drug glycosylation modification and stability improvement among batches are achieved, scientific innovation and industrial practicability are achieved, and a technical foundation is laid for high-quality production of next-generation antibody drugs.

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).

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.