The Smart Choice

Monoclonal antibodies are a well-established target molecule in the biopharmaceutical industry landscape and can treat an extensive range of conditions. Today, their production is well understood and generally easy to scale. However, the biopharma innovators aren’t resting on their laurels when advancing the science behind these therapies. Antibody fragments are among the most promising formats, improving delivery to target sites and increasing binding affinity. Moreover, progress in engineering allows the recombinant production of antibody fragments at economically viable titers.

As antibody fragments are relatively new technologies, proper analysis and purification make all the difference in providing safe and efficient therapies to the patients. Our biopharma partners do not only rely on Tosoh Bioscience’s high-quality chromatography solutions; they also trust our experts to enable them to develop the most cost-effective processes.

Easing the burden

Biopharma manufacturers constantly aim to produce a high concentration product at the antibody capture step so that all subsequent operations, including the polishing step, are as cost-effective and time-efficient as possible. This was historically a challenge for fragments, as they cannot bind to traditional resins like Protein A (which relies on Fc binding). Protein L is now the first port of call for such purification challenges. The proteins on the surface of the Protein L resin matrix bind to antibodies via their kappa light chains (present in many antibody fragments) to remove impurities. Though well suited for the task, Protein L is a much younger medium when compared with its counterparts – and developers report that traditional resins often exhibit a lower dynamic binding affinity. Simply put, only low quantities of the target molecule can be loaded onto a column, thus limiting its performance, resulting in a more costly and time-consuming antibody capture step.

Another potential hurdle for manufacturers is the low chemical stability of the resin over the repeated cleaning cycles. After a column has run, it is usually cleaned with an alkaline solution. However, the poor chemical stability of traditional Protein L resin often resulted in a partial loss of performance. To overcome this issue, the resin needs to be exchanged after a low number of cycles – leading to increased costs and workload!

To address those challenges, Tosoh has developed a leading-class alkali-stable Protein L called Toyopearl AF-rProtein L-650F. First and foremost, it has been designed with high dynamic binding in mind, which means that more protein can be loaded onto the column and that fewer runs are needed to purify the same amount of target molecule. In fact, our Protein L affinity resin’s dynamic binding capacity is at least two times larger than other commercial products for Fabs at any residence time, which clearly helps optimize manufacturing efficiency and productivity. We have investigated not only the performance of the antibody fragment capture step but also the reusability of the resin during a whole purification campaign. Thanks to its improved alkaline stability, more purification cycles can be run on the same column. Lastly, our extensive experimentation with different washing solutions and our expertise in packing helps improve product purity and resin lifetime. Down the line, our biopharma partners need less resin, which allows them to develop the most effective processes, and they rely on our expertise to help them develop their processes as fast as possible.

The science behind the success

Though our Protein L is helping to revolutionize the capture of antibody fragments, we recognize that sustained improvement is mandatory – and, for us at Tosoh, that means a continued focus on high-quality research and development. Indeed, we are committed to several extensive research programs – both internally and with our partners - that aim to optimize overall manufacturing productivity. For example, we are currently evaluating how a Protein L mediated capturing step can be transferred onto a continuous system. We expect that this productive, efficient option will expand possibilities for our customers.

Ultimately, Tosoh is dedicated to developing increasingly efficient solutions for the purification of antibody fragments. With an established history and wealth of technical expertise, I believe we are the best partner for drug developers looking for cost savings and increased productivity.

Resolving Problems

Wacker Biotech bundles WACKER Group’s biopharmaceutical activities. The company has years of experience under its belt when it comes to the production of antibody fragments, using E.coli as an expression system. But the process is not without its challenges. Here, Ilona Koebsch, Business Development Manager, and Thomas Walther, Expert Downstream Processing at Wacker Biotech outline some of the manufacturing challenges the company has faced – and how Tosoh’s support fed into the solutions.

What are the most interesting antibody fragment subclasses?

Koebsch: Antibody fragments differ in terms of size, structure, and function. But Fabs – and all variants containing antigen-binding sites – represent the most interesting antibody fragment technologies from a therapeutic point of view. Notably, the smaller size of the fragments permits penetration into tissues inaccessible to fullsize mAbs – and that’s essential in many therapeutic and immunohistochemical aspects. In addition, these fragments can be more easily and cheaply manufactured using prokaryotic expression systems, such as E.coli, because of their relatively small size.

What are the main challenges in the production of Fabs?

Walther: Depending on the structure, Fabs can be soluble or can assemble into inclusion bodies (aggregated recombinant proteins). Manufacturers must also maintain their folding and structure during fermentation and downstream processes to ensure the Fab is biologically active. For upstream processing, the main task is always to find the best combination of the expression system (host and plasmid) and cultivation parameters to achieve an efficient but robust manufacturing process. But, in my view, the most critical challenge is finding appropriate models to simulate certain outcomes or to mimic large-scale processes on a laboratory scale.

In downstream processing systems, the main challenges relate to the complexity of the sample matrix, development times, and the low tolerance of affinity resin against many common cleaning solutions.

What is WACKER’s approach to tackling the challenges?

Koebsch: WACKER has developed two unique E. coli based expression systems enabling highly efficient production of different antibody fragments. ESETEC® is a unique expression system that allows for the controlled secretion of correctly folded recombinant protein products in a fermentation broth. In short, it helps simplify primary recovery and purification processes which brings down cost-of-goods.

For hydrophobic antibody fragments, we offer FOLDTEC® technology, which consists of highly efficient E. coli producer strains, an antibiotic- and phage-free plasmid maintenance system, and comprehensive refolding know-how.

We do not have a proprietary technique to overcome issues in downstream processing. Chromatography resins with high capacities, good resolutions, low back pressure, easy handling (in terms of column packing), short delivery times in combination with a long shelf life, and low nonspecific binding behavior are all needed!

How did Tosoh support WACKER tackle those challenges?

Walther: On the recommendation of a colleague at another WACKER site, we used the Tosoh AF-rProtein L-650F resin in a project where our preferred resin failed due to a very low binding capacity.

We compared Tosoh’s resin against three others that claimed to support Kappa light chain affinity binding. The AF-rProtein L-650F resin showed a significantly higher binding capacity (around three times higher) than the competitors, as well as good recovery of the target protein. The number of proteinogenic impurities was also the lowest of the tested resins.

Finally, AF-rProtein L-650F exhibited better flow rates than the other tested resins, which allowed us to use smaller column volumes to house the resin – with real cost advantages for our customers. We also had great support from Tosoh’s packing and technical sales specialists, who made recommendations for the best types of columns to use with their product.

It was apparent to us that Tosoh’s Protein L resin is a strong competitor – and it should make other suppliers rethink their current product lineups.