Bispecific antibodies (BsAbs) are artificially engineered immunoglobulins that can simultaneously bind two distinct epitopes or targets. Their dual-targeting capacity renders expanded therapeutic potential, especially in the area of oncology and autoimmune diseases. Currently, there’s a strong interest in the design, production, and purification of BsAbs to harvest improved efficacy through new mechanisms of action.
Chromatography is one of the most commonly used means to purify antibodies. The isolation and purification of BsAbs is the concentration of a specific antibody or antibodies and the removal of non-specific antibodies and contaminants. As a professional antibody manufacture supplier in the world, Creative Biolabs has established a series of affinity purification methods, including Protein A chromatography, to handle expectations for more mature antibody production. We’d like to help clients to obtain high-quality BsAbs with higher purity, lower costs, and less time through multiple effective platforms.
Early BsAbs were made by Quadroma technology, involving the somatic fusion of two hybridoma cell lines, or by chemical conjugation technologies. Both of these approaches posed significant manufacturing and purifying challenges. The expression of three chains leads to three products: the desired heterodimer bispecific molecule and two parental homodimers (as product-related impurities). Hence, efficient purification steps are required to purify the BsAbs from the undesired antibody molecules that might have similar biophysical characteristics as the desired BsAbs.
Fig.1 Diagram illustrating bispecific (FcFc*) and related contaminating antibodies (FcFc, Fc*Fc*) synthesized in the production bioreactor. (Tustian, 2016)
The additional steps in purification would cause either a decrease in yield or purity of BsAbs. Large scale purification of BsAb molecules needs robust, preferably a single-step purification method. Recently, a novel purification development aspect of bispecific formats has been described, based upon commercially available GMP-quality resins, for example, staphylococcal Protein A. Through this approach, purification of the bispecific is facilitated by a substitution of two amino acid residues (named the star substitution) in the CH3 domain of one of the heavy chains. This allows the isolation of the bispecific dimers via selective elution from a Protein A column.
Fig.2 Flow scheme of suggested purification processes for star substitution containing bispecific antibodies exhibiting (A) and not exhibiting (B) VH domain SpA binding. (Tustian, 2016)
The characteristics of BsAbs offer them advantageous over conventional monoclonal antibodies. They recognize two antigens on a single receptor that can trigger different inhibitory mechanisms against the single target. In other instances, BsAbs targeting two pathogenic mediators can block two signaling pathways at the same time, enabling additive or synergistic effects.
The production of BsAbs usually results in lower total yields and less standardized purification protocols. At Creative Biolabs, our expert groups provide a diversity of valid affinity purification methods to capture target BsAbs as many as possible. As well, we’re pleased to propose not just one possible strategy to remove commonly found impurities or contaminants in the antibody production. Our flexible and effective BsAb purification services contain items including but not limited to:
Affinity chromatography depends on the reversible interaction between a protein and a ligand immobilized in the chromatographic matrix. The material is applied under conditions that favor specific binding to the ligand as the result of hydrophobic and electrostatic interactions, Van Der Waals' forces, and hydrogen bonding. After washing away the unbound molecules, the bound proteins are recovered by altering the buffer to those that favor desorption. This technique has been used not only to isolate antigen-specific antibodies but also to separate certain contaminants from biological samples.
Here, we have developed a single-step, Protein A chromatography process for BsAbs purification in early screening. This method is to separate the BsAb from fragments, like individual heavy and light chain pairs. By applying a pH gradient elution from pH 5.0 to 3.5, most of the fragments can be wiped off as early as the capture step, without the need for additional protocols. With the help of us, BsAb constructs are efficiently purified to show more than 90% purity and 85% yield in the downstream process.
There are also some other immunoglobulin-binding bacterial proteins, such as protein G and protein L, that have been wildly used in the purification and detection of bispecific antibodies.
Protein tag refers to a protein or polypeptide that is fused and expressed with a target protein during the preparation of recombinant proteins. The role of a tagged protein is generally to promote the solubility and stability of the target protein. With the advancement of research and the development of technology, various labels have emerged and play parts in BsAb isolation and purification. Notably, for BsAbs without Fc regions, Bispecific fragments, Creative Biolabs offers chromatography methods for different labels, including His-tag, Flag-tag, Avi-tag, GST-tag, Sumo-tag, HA-tag, c-Myc-tag, etc. These labels could increase the output of purified BsAb products and meet different experimental purposes in antibody purification protocols.
BsAbs can be designed in many different formats and each category has its own benefits. They’re broadly divided into two categories: IgG-like Fc-containing proteins and smaller entities without Fc regions. In general, IgG-like Fc-containing BsAbs production is technically more complex than their Fc-free counterparts.
Due to the co-expression of up to four different polypeptide chains or assembly involving chains with extended length, the production of recombinant IgG-like BsAbs is often accompanied by an increased level of product-related impurities (e.g. aggregates and byproducts). Therefore, we have introduced several useful methods that can largely remove product-related contaminants in IgG-like BsAb purification. In the below, homodimer-removing approaches from Creative Biolabs for unmodified, engineered BsAbs are summarized.
|Common BsAb Specific Byproducts and Methods For Their Removal|
|Homodimer||Protein A chromatography, Ion exchange (IEX) chromatography, Hydrophobic interaction chromatography (HIC), Hydroxyapatite (HA) chromatography, Multicolumn countercurrent solvent gradient purification (MCSGP), Dual anti-idiotypic purification|
|Unmodified BsAbs||Cation exchange (CEX) chromatography, Anion exchange (AEX) chromatography|
|pI engineered BsAbs||Ion exchange (IEX) chromatography|
|Affinity engineered BsAbs||Protein A chromatography (with modified Protein A binding avidity)|
|κλ-body||A robust purification process consists of three affinity steps, involving MabSelect SuRe, KappaSelect and LambdaFabSelect resins|
|Half and ¾ antibodies||Hydroxyapatite (HA) chromatography, Protein A chromatography, Hydrophobic interaction chromatography (HIC), Cation exchange (CEX) chromatography|
|HC and LC (monomer or dimer)||IEX chromatography, Hydrophobic interaction chromatography (HIC), Mixed-mode|
|Aggregates||Hydrophobic interaction chromatography (HIC), Hydroxyapatite (HA) chromatography, Capto adhere, Capto MMC ImpRes|
➢ There’re numerous affinity chromatography methods that can be provided for BsAb purification.
➢ Using the single-step process, desired BsAbs can be purified and collected with high efficacy and recovery.
➢ Our technical platforms can effectively remove product-related impurities in IgG-like BsAb purification.
➢ Advanced technologies, dedicated devices, and experienced experts guarantee a reliable outcome.
There is increasingly attractive in the production of BsAbs to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been constructed and are currently under development. As a top-ranking provider in the antibody market, Creative Biolabs offers various different chromatography tools to deal with the urgent demands for more purified antibody modalities. If you want to know more information about our BsAb purification services, please don’t hesitate to contact us.
1. Tustian, A.D.; et al. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity. MAbs. 2016, 8(4): 828-838.