Producing insoluble protein in inclusion bodies can and often is more economical from a bioprocessing perspective. We explain the pros and cons of soluble and insoluble processes, share our experience with isolating, solubilizing and properly re-folding, and argue that the right approach to insoluble processes can not only be viable at scale but can offer great opportunities for yield improvement versus soluble production.

A transformative era in biopharmaceuticals

The biopharmaceutical market has experienced incredible growth over the last several years, driven by new scientific discoveries and technological advancements. As a result, a wide range of novel modalities are emerging during this transformative era, bringing not only new opportunities but also new challenges.

Finding more economical options

While mammalian cell culture is still a dominating platform for recombinant protein production, most therapeutic protein classes are made in microbial systems. The bacterial cell used in this expression system could offer four production systems in one: expression in the cytoplasm or the periplasm or extracellular secretion, i.e., expressing in the soluble form, or inclusion bodies (IBs), i.e., the insoluble form.

Despite the historically bad reputation of the latter, IBs can actually often present a more economical option from a bioprocessing perspective. Knowing how to use them effectively and efficiently, though, requires extensive experience with microbial processes using advanced engineering and process development capabilities. 

Q&A with Lonza’s experts

Lonza's Jonas Mueller, team manager, and Nikolay Krumov, senior project leader, recently hosted the webinar Inclusion Bodies ― Mother Nature’s Help or Hindrance? In it, the pair discussed the pros and cons of soluble and insoluble processes, shared their experiences with isolating, solubilizing, and properly refolding, and argued that the right approach to insoluble processes can not only be viable at scale but also can offer great opportunities to improve the total process outcome versus soluble production. They also reviewed strategies and future innovations to improve the performance of Lonza’s IB processes. The following Q&A session was held afterwards, where Mueller and Krumov addressed questions from attendees about the information provided during the presentation. 

The insoluble route, especially refolding, is known for requiring large volumes. What is the largest volume refold process your team has run, and how do you deal with the resulting waste streams?

Mueller: Lonza’s facility in Visp, Switzerland, includes two 15,000-liter stainless steel fermenters in two separated lines with 23,000-liter tanks for solubilization and refold, which have been successfully used in parallel to facilitate these unit operations.

In regard to waste, we follow the same approach described for scale-up in general, which is that, as soon as the process is fixed, small-scale samples are provided to the safety and environmental experts for analysis and a suggested waste treatment plan. 

Based on these results, we assess whether it can be properly managed in the future. This allows any changes to be made should issues be detected at this early phase. 

Solubilization and refolding require harsh buffers. How does Lonza prepare buffers at a large scale?

Mueller: The buffer conditions are a challenge; however, all of Lonza’s facilities are equipped to manage these buffers for the targeted scale. For large scale, we have dedicated buildings and buffer prep facilities capable of preparing the amount of buffers at the right conditions and in the amounts needed. 

If a molecule is expressed as an inclusion body, does Lonza have the technical capability to change expression to a soluble process?

Krumov: When we start to develop a process, we explore all options, including changing the host. Often, though, the molecule will choose the most effective path for expression, which is something we see in the early phases of development. Some projects have expression in inclusion bodies while others are soluble. If the soluble path is viable, we focus on increasing yield and productivity by using a variety of tools, including strain optimization. 

What are typical process yields of a soluble and IB process, and what approaches do you take to improve this?

Krumov: Yield is an important parameter during process development. When we deal with soluble processes, we expect to see an overall yield of around 30%; for IB processes, overall yield is around 15% and below, due to more unit operations and longer processes impacting the yield. 

However, yield is not the main target but rather how much product we get at the end of the process, which depends on other specifics, such as titer and even bottlenecks in the process. For example, if we have a soluble process with one gram per liter titer at the end of the fermentation and an overall yield of 50%, the end result will be 0.5 gram per liter of product.

Conversely, if we have an inclusion body process with 10 grams per liter titer, which is a common titer for IB processes, and a yield of 10%, the end result will still be one gram per liter of product. Therefore, it is important to find balance and see what the most important metrics are.

Does Lonza freeze IBs?

Krumov: Yes, we’ve frozen IBs in the past quite often during product development because it’s a great opportunity to save both material and time. 

However, in manufacturing, we believe that continuous manufacturing is definitely a better way to go. One of the reasons we believe this, which also ties nicely to the data presented in the webinar, is because, as we’ve seen during process development, consistency issues with IBs can worsen during freezing. This, in addition to ensuring reproducibility and robustness when dealing with large amounts of IBs, can be challenging.

Therefore, our facilities are set up for our strategy of feeding one upstream batch into one downstream batch. In addition, we often suggest to customers with lower product demand to use our smaller or differently-sized facilities rather than freeze IBs. 

Can Lonza’s Visp facility support pegylation?

Mueller: Yes, we can support pegylation and development of processes involving pegylation.

Is solubilization an inconvenient process?

Mueller: From a manufacturing point of view, it can be inconvenient. Huge volume increases are often required and buffer conditions can be quite harsh for this step. However, it is possible to overcome these challenges if your facility is equipped accordingly, and you plan for them ahead of time. This is a great example of where the benefits of Lonza’s development process really come into play because we always double check that the process we develop at small scale, especially in respect to solubilization and refold volumes, fits the tanks that are available at scale in order to avoid issues later.

Scale-up can be problematic during the fermentation process. Can you have enough aeration efficiency, even in a 15,000-liter fermenter?

Krumov: Yes, our facility can handle high cell density fermentations, including the required oxygen supply. When we develop a process, we ensure it is done with all of our production facility capabilities in mind. 

Can you elaborate on Lonza’s high-throughput methods for the development of refolding conditions as discussed in the webinar?

Krumov: When we develop IB processes, we also have to identify solutions for solubilization and refolding, which we do using design of experiments, as this allows us to be more efficient when screening parameters. However, this approach requires high-throughput analytics that give us the basis to evaluate those parameters, which can be a limiting factor. 

Can IB purity be affected negatively by the homogenization process?

Mueller: In our experience, we have seen it be both positively and negatively affected. We have had examples where the homogenization process improved the IB purity while, in other cases, the lysis of the cells through homogenization led to unfavorable conditions because everything became uncontrolled. 

A clear advantage of IBs is that, as long as they are in IB format, one can also consider adding multiple IB washes to address purity questions. This ties back to what has been stated before, which is that, often, the solution must be tailored to the unique needs of a specific process.

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