Common Problems and Solutions in Recombinant Antibody Mammalian Expression Experiments

Q1. Under what circumstances is it appropriate to use the lactation system for antibody recombinant expression?

A1. The essence of antibodies is immunoglobulin. When expressing eukaryotic proteins, the corresponding expression system will be selected according to different eukaryotic protein expression needs. The mammalian cell protein expression system can provide a cell environment similar to the natural cell environment, suitable for the expression of complex eukaryotic proteins. The mammalian protein expression system can synthesize proteins with structures and functions similar to human proteins. Recombinant antibody expression by the mammalian cell protein expression system can ensure correct post-translational modifications, such as glycosylation. The mammalian cell expression system can express a large amount of proteins, which is suitable for large-scale production of recombinant antibodies. In addition, proteins expressed by mammalian cells can be secreted outside the cell, making the purification process of recombinant antibody expression simpler. Based on the powerful post-translational modification function of mammalian cell expression systems, mammalian protein expression is also closer to the structure of human derived proteins. Therefore, mammalian expression systems are commonly used for therapeutic antibody development.

Q2. How to analyze the protein sequence of a given antibody?

A2. Firstly, based on the protein and species that the customer wants to express, the corresponding protein information is searched to find its amino acid sequence. Then transmembrane analysis, signal peptide analysis, and hydrophilicity hydrophobicity analysis are performed. If the protein contains a signal peptide region, the signal peptide region needs to be removed during expression. When there is a transmembrane domain in the protein, insect expression systems are generally used, which can cross up to 7 times. By observing the peaks generated in the analysis of hydrophilicity and hydrophobicity, it is possible to determine whether the protein as a whole is hydrophilic or hydrophobic. If it is a hydrophobic protein, SUMO tags need to be added to promote solubility. After obtaining the above relevant information, subsequent selection of expression systems, enzyme cleavage sites, tags, and vectors can be carried out.

Q3. How to design recombinant antibody expression?

A3. The principle of recombinant protein expression is mainly to clone the target gene into an expression vector, transform it into the host cell, and then produce the target protein through transcription and translation processes. In the production of antibodies, commonly used expression vectors include plasmids and viral vectors, in which promoters, transcription stop signals, etc. can be inserted to ensure efficient transcription and translation of genes. Inserting the gene of the antibody into the appropriate position of the vector, and then transfecting the constructed vector into host cells, stable expression cell lines of the antibody can be screened using labeling for large-scale recombinant antibody preparation. The commonly used antibody expressing mammalian cells include CHO cells and HEK293 cells. Adding His tags, FLAG tags, etc. is beneficial for affinity purification of antibodies.

Q4. What are the commonly used methods for mammalian cell transfection?

A4. When performing mammalian cell transfection, transient transfection or stable transfection are mainly used. Instant transfection is a simple operation that can express the target gene in a short period of time, and is commonly used for small-scale protein synthesis in the short term. Stable transfection can be used for long-term experimental research and the development of mammalian cell antibodies. The main methods of cell transfection include physical transfection (electroporation, microinjection, gene gun, etc.), chemical transfection (liposomes and their substitutes, calcium phosphate, etc.), and biological transfection (various viruses, including adenovirus, lentivirus, retrovirus, adenovirus, etc.).

Q5. What should be noted when conducting mammalian cell culture?

A5. During the process of cell culture, it is important to pay attention to aseptic operations to prevent contamination of the cells. When reviving cells, the opening of the cryotube should not touch the water surface to reduce the possibility of contamination by impurities in the water. When carrying out cell passage, the time for trypsin digestion should be adjusted according to the cell type and cell state. When cell recovery and freeze-thaw, attention should be paid to "slow freezing and rapid melting" to reduce temperature damage to cells. When cultivating cells, the status of the cells should be checked daily. When the cells grow well and the density is moderate, passage can be carried out.

Q6. What are the common types of cell contamination? How to prevent it?

A6. According to different sources of pollution, cellular pollution is mainly divided into physical pollution, chemical pollution, and microbial pollution. Both supercooling and overheating can cause changes in cell state, even leading to cell death. Exposure of cells to radiation can damage their DNA structure, resulting in cell death or genetic variation. Impurities or concentration issues in components such as culture medium, water, and reagents can all affect cell growth and even have toxic effects. The use of substandard reagents during cell processing can also affect cell growth. Microbial contamination is a relatively common and serious contamination in the process of cell culture, which can be divided into bacterial contamination, fungal contamination, mycoplasma contamination, and viral contamination according to the type of microorganisms. Once cells are contaminated, it will affect their state, prolong the cell culture cycle, and affect experimental results. Therefore, the cell culture environment should be kept clean and tidy, and relevant regulations should be strictly followed during the operation process to ensure aseptic operation.

KMD Bioscience has established a comprehensive recombinant antibody mammalian expression system, including but not limited to FreeStyle 293-F cell line, Expi 293-F cell line, Expi-CHO-K1, and Expi CHO-S cell lines. Combined with KMD Bioscience's designed mammalian expression vector (containing a full-length CMV promoter and optimized secretion signal peptide sequence), it can provide customers with higher expression levels of recombinant antibody mammalian cell expression and antibody development services. In addition, KMD Bioscience has a mature vector cell transfection efficient expression system, which can shorten the experimental cycle and save customers time.

Reference

[1] Dean DA, Gasiorowski JZ. Liposome-mediated transfection. Cold Spring Harb Protoc. 2011;2011(3):prot5583.

[2] Philippeos C, Hughes RD, Dhawan A, et al. Introduction to cell culture. Methods Mol Biol. 2012;806:1-13.

[3] Zhang R, Rao M, Li C, et al. Functional recombinant human anti-HAV antibody expressed in milk of transgenic mice. Transgenic Res. 2009;18(3):445-53.