Optimizing Antibody Production in E. coli

Antibody Expression Landscape

The history of therapeutic production has been dominated by the testing and production of antibodies in mammalian tissue culture, most notably using CHO cells (derived from Chinese Hamster Ovaries). 

The demand for new and improved antibody production systems has arisen in recent years.  Many researchers have been working towards alternative antibody expression systems such as yeasts (Saccharomyces or Pichia), filamentous fungi, transgenic plants, and E. coli. 

Use of E. coli expression systems has been limited mainly to the production of antibody fragments.  Fab (antigen binding fragment) fragments are the portion of the antibody responsible for binding the target antigen.  These fragments have been successfully expressed and secreted in E. coli.  Fabs are frequently used in diagnostic applications, therapeutics, and in testing variable regions slated for reincorporation into full-length monoclonal antibodies.  Another successful application in E. coli production is the fusion of a functional protein with a Fab. The antigen targeting-specific Fab region is fused to a functional protein sequence.  Creating targeted therapeutics with enhanced cell killing is one application of this approach.

Other strategies involving antibody fragments include, fusing target specific protein domains such as receptor fragments to Fc (receptor-binding fragment) regions.  The Fc fragment of the antibody is responsible for the long serum half-life along with activation of the immune system.  Applications of Fc fusions depend on combining the binding activity of the fusion partner with the activation of the Fc region.  However, production of the Fc region in E. coli can be problematic due to the difficulty of effectively expressing the Fc fragment in bacteria.  This may explain why production of the full monoclonal antibody in E. coli has also remained an elusive goal.  As described below, CODA has identified improved methods for expression of both fragments and full monoclonal antibodies in E. coli. 

Antibody Expression in E. coli

It is well known that E. coli has many desirable advantages.  The extensive experience with E. coli-based protein expression and the ease of genetic manipulation in E. coli makes it a highly attractive host for expressing antibodies.    The chief limitation has been unreliable protein expression levels and difficulties in consistently secreting the active antibody proteins. 

Conventional production systems are also time and labor-intensive.  It takes approximately four to six months to produce a stable production system in a mammalian system as compared to one month in E. coli. While the mammalian cell machinery is crucial for antibody production attributes such as glycosylation, many opportunities exist for an effective E. coli antibody production system.

E. coli’s greatestimpact thus far has been in the area of antibody development utilizing phage display systems.    Using this technology, it is possible to screen astronomical numbers of sequences to find binding regions that are highly specific and bind tightly to selected targets.    Typically, once these improved binding regions have been optimized, the entire region is grafted back into full-length antibodies for expression in mammalian systems.  At this stage, the ease of manipulation in E. coli is lost.

Using Translation Engineering™ to Overcome Limitations

CODA has demonstrated the utility of using Translation Engineering to optimize genes for expression of antibodies and antibody fragments effectively in E. coli.  Translation Engineering includes industry standard techniques such as removing rare codons and smoothing out RNA secondary structure.  In addition, CODA has identified translation pause signals that effect the step-wise kinetics of the ribosome while it is translating the antibody mRNA.  Translation Engineering is unique in that CODA can manipulate these translational pauses to perform proprietary optimizations on the antibody constructs.  The redesigned gene construct is then put into an appropriate vector which may include both heavy and light chain components.  In addition to applying these techniques to Fab commercial scale-up, CODA has been able to express an active, full-length monoclonal antibody in E. coli.

Manipulation of Fab regions in E. coli has been widely used in creating fusion proteins.  However, many research groups report difficulty in producing Fab and Fab fusion proteins in E. coli at commercial scale.   

Case Study

A pharmaceutical company was struggling to scale production of a fused Fab and toxin expressed in E. coli.  During optimization of the Fab binding and functionality, small amounts of Fabs could be produced for testing and development purposes.  During scale-up for clinical trials, the economics of production and purification were not acceptable for the final therapeutic application.  CODA optimized genes (open reading frames) for both the heavy and light chain portions of the Fab using Translation Engineering.  Working with CODA, the client was able to achieved improved yield and now has a working solution in large scale GMP production.