Antibody production plays a fundamental role in drug discovery. Understanding its process and the factors affecting the production of antibodies is the first step in ensuring you obtain high-quality antibodies for your research. In this article, we explore these factors and how they play a part in antibody expression, production, and reformatting.
Antibody Sequence and Structure
Antibodies are a type of protein molecule known as immunoglobulins that play a significant role in immune system function. Four fundamental polypeptides create the antibody structure. These four polypeptides consist of two heavy and two light chain sequences that create a Y-shaped particle.
The tips of the Y-shaped structure, which are made up of the variable regions of the light and heavy chains, contain amino acid sequences that determine the specificity of the antibody for a particular antigen. The constant regions of the heavy chains determine which portion of the antigen is recognized by the antibody.
There are five isotypes of antibodies: IgM, IgE, IgA, IgD, and IgG. Each type of antibody has a different constant region that determines its function in the immune system.
Expression Vector Design
Stability is a significant factor affecting antibody production due to the possibility of aggregation. The structure, sequential format, and expression design play a role in creating a stable culture. Choosing a feasible expression vector is essential in optimizing the throughput and stability factors.
Chinese hamster ovaries (CHO) cells are commonly used in the production of proteins and antibodies due to their stability. This is because cells with dual promoter systems offer design efficiency and provide stable environments for expression, leading to high levels of antibody production.
Host Cell Selection
The host cell selection process is vital in ensuring adequate throughout and proper expression. There are several host cells available, each serving a fundamental purpose per the antibody of interest. CHO cells and human embryonic kidney 293 (HEK293) cells are two of the most commonly used hosts because they offer optimized stability and expression for recombinant antibodies of mammalian origin.
HEK293 works well in small-scale transfection production orders. If necessary for large-scale orders, labs will use PEI (polyethyleneimine) and calcium phosphate.
CHO cells are preferred for mammalian production lines, with over 70 percent approval for CHO monoclonal antibodies worldwide
It’s worth noting that Bon Opus Biosciences will use Epxi293, EpxiCHO, and E. coli for host cell selection processes and will collaborate with you to make the most feasible selection for the order.
For antibody production, stable or transient mammalian cells are required, and the culture conditions play a crucial role in achieving the desired outcomes of the production process. CHO offers optimal stability, but HEK can achieve a high transfection rate. The culture’s conditions are closely related to the production process outcomes and the time scale for the production process. HEK293 will work best for transiently expressed antibodies, with a rapid production phase, and can be used with any mammalian cell.
In stably transfected culture conditions, the host and foreign cells combine to create a replication. Stable transfection begins with a transient phase, but through amplification, antibody clones form and serve large-scale production yields best. The cell and culture can sometimes experience stress when a selection marker is introduced with the expression vector. If this occurs, labs may conduct a dose-response test to understand suitable concentration outcomes.
Regardless of host cell selection, most cell pathways will require additional media introduction to the culture for optimal growth rates. This additional media helps with proliferation and optimizes the transient expression of the antibody of interest. Typical media used in this culture include growth factors, hormones, and signaling substances. The selected media will be carefully considered per the yield requirements and antibody intentions. Labs conduct media analysis tests to determine the best growing mediums, and this process also highlights which additives will encourage the antibody expression without altering cell practicality.
Purification is a crucial step in antibody production that impacts the yield and quality of the final product. Various purification methods are available, and each will work for specific applications and requirements.
Size Exclusion Chromatography (SEC), or Gel Filtration, is a common antibody production purification method. This process involves a molecular separation process to enable resin pass-through based on their size. The molecules range in size when separated, and the goal is to be able to isolate one molecule for identification and analysis. The sample will indicate the overall molecular weight that passes through.
IEX, or Ion Exchange Chromatography, is another common antibody purification method that helps separate antibodies based on their net surface charges. Every antibody of interest has a distinct net surface charge and pH balance Antibodies are loaded onto a column packed with a charged resin, and the different antibodies are separated based on their affinity to the resin under specific pH and salt conditions. IEX is effective for purifying antibodies with different surface charges and isoelectric points.
Zone Electrophoresis, or ZE, is a less common antibody purification method with proven responses throughout the biology sphere. This involves immersing the cells into a specific solution designed to highlight the charge-to-mass ratios of molecules. From here, they are separatable based on size, and the many distinct characteristics can help create the designated zones, also known as bands.
Bon Opus Biosciences specializes in recombinant antibody production processes with a transparent and affordable approach to antibody reformatting and much more. Learn more about our services and custom solutions through various completed case studies, or request a quote for service today.