一. The expression process of recombinant antibodies in mammalian cells?

The traditional way to produce antibodies is to stimulate the animal immune system with antigens. This method requires the preparation of antigens first, and it is difficult to produce antibodies in large quantities using this method (it requires a lot of experimental consumables and time). The method of producing antibodies in mammalian cells has certain advantages. ① The gene sequence of the antibody is known, the sequence is cloned into the expression vector, introduced into the mammalian cell body for culture, and a stable cell line that can stably produce the antibody gene is finally obtained through a series of screening. This process requires a lot of time to screen out a cell line that is sufficiently stable in expression and can be stably produced in subsequent experiments for a long time. ② For the mass production of antibodies with unknown sequences, first stimulate the animal immune system to obtain a small amount of antibodies, obtain the antibody sequence through sequencing, and screen the stable expression cell line through the expression of recombinant antibodies in mammalian cells to meet the needs of mass production.

二. When expressing recombinant antibodies in a lactation system, what are the differences between HEK293 cells and CHO cells?

The advantages of CHO cells are:

(1) Able to grow stably in chemically defined and serum-free suspension media;

(2) The cell genome information is clear and it shows reasonable safety in response to human pathogenic viruses;

(3) Able to express human-like post-translational modifications;

(4) It is easy to obtain genetically modified cells.

The advantages of HEK293 cells are:

(1) Has a faster growth rate;

(2) It has higher growth density and high transfection efficiency;

(3) Post-expression modification is closer to the structure of human proteins.

三. How to improve cell growth and anti-apoptosis abilities?

To improve the growth capacity of cells, anti-apoptotic genes can be overexpressed. For example: overexpression of Bcl-2 in NS0 and CHO cells can inhibit the mitochondrial apoptosis pathway; overexpression of P21 and P27 in CHO cells can arrest the cell growth cycle in the G1 phase; through overexpression of E1B-19K, Aven genes, or Heat shock proteins, taurine transporters, etc. can also significantly improve cell activity and extend the culture period.

In order to reduce by-products during cell culture, key genes in the metabolism of by-products are manipulated. For example, by up-regulating the cellular pyruvate carboxylase gene and down-regulating the lactate dehydrogenase gene, the production of lactic acid is reduced; by over-expressing urea cycle enzymes, carbamoyl phosphate synthase, ornithine carbamoyltransferase, etc. to reduce ammonia production.

四. How to improve the translation, modification and secretion capabilities of recombinant antibodies?

For most recombinant cells, translation, modification, and secretion are the rate-limiting steps of the antibody biosynthetic process. Therefore, molecular chaperones involved in the biosynthesis of the target protein can be genetically manipulated to improve the recombinant antibody expression ability of host cells. Such molecular chaperones include: protein disulfide isomerase, endoplasmic reticulum oxidoreductase, the heavy chain-binding protein, ERp57, GRP94, X- Box-binding protein 1 (XBP-1S), etc.

The glycosylation modification of antibodies has a significant impact on their biological activity. Using cell engineering technology to modify the glycosylation modification ability of the host can improve the clinical efficacy of recombinant antibodies. For example, by knocking out fucosyltransferase (FUT8) or overexpressing acetylglucosaminyltransferase (GnT-III), the fucose content in the recombinant antibody sugar chain can be reduced to improve the ADCC effector function of the antibody drug; By overexpressing sialyltransferase, the sialic acid content of the antibody is increased to improve the anti-inflammatory activity of the antibody.

五. What are the advantages of recombinant antibodies?

(1) Short cycle: recombinant antibodies can be produced in a few weeks, while hybridoma antibody preparation takes several months;

(2) High consistency and good repeatability: the genes of recombinant antibodies are determined, and antibody production is controllable and reliable, which makes the difference between batches of antibodies small and the repeatability good;

(3) High sensitivity and specificity: antibodies selected by genetic engineering and recombinant technology are of higher quality;

(4) Low immunogenicity: no immunization and screening are required;

(5) Large-scale antibody preparation: animal-free in vitro production can be achieved.

六. How to improve the stability of antibodies?

Optimize elution conditions: gradually reduce from high pH; neutralize purified samples in time; prepare and store samples at low temperature during purification experiments; add protein protectants, such as glycerol and Tween; dilute antibodies in time when the concentration is high to avoid high concentration aggregation.

Recombinant antibody production makes it possible to prepare humanized antibodies and humanized antibodies. It is a key technology for the industrialization of monoclonal antibody drugs. Recombinant antibodies are widely used in immunodiagnosis, therapeutic antibodies and other fields. TekBiotech has a complete recombinant antibody expression production platform and rich experience in recombinant protein and recombinant antibody expression. Based on phage display technology, we can provide customers with high-quality recombinant antibody preparation and screening services. At the same time, combined with a large-scale recombinant protein fermentation platform, we can produce monoclonal antibodies including single-chain antibodies (scFv), single-domain antibodies, humanized antibodies and Fc chimeric antibodies for customers. TekBiotech's transient expression system uses CHO cells and HEK293 cells as expression hosts for suspension culture, and can produce milligrams to kilograms of recombinant antibodies. In addition, we can perform customized antibody preparation according to your needs, including tag removal, endotoxin control, SEC-HPLC analysis, glycosylation analysis, etc.