Enzymes are in growing demand as drug substances for antimicrobials, anticoagulants, and enzyme replacement therapies for a range of disease areas. Enzyme manufacturing processes come with a unique set of challenges — for example, unlike antibody manufacturing, it is more challenging to develop a platform production process that can be applied to many different enzymes. Because every enzyme is unique, it is often necessary to establish a unique, tailored process for each.
Enzyme production must be designed to balance high yields with high purity from an economically viable process. Developers might not recognize that processes that work in-house or in a small-scale lab environment can be difficult to transfer to a contract manufacturer, and some just aren’t feasible for scaled-up manufacturing.
Many companies also don’t perform an in-depth build versus buy analysis during development. As a result, in the early days it can seem to make sense to carry out as much as possible in-house. Eventually, if their business model is successful, companies can wind up victims of their own success. They may reach a point at which outsourcing is preferable for quality and outscaling reasons — but by that point, demand catalyzes pressure on their end and timetables need to be short to advance to larger-scale production.
It is easy to underestimate the time it can take to successfully develop or transfer processes to a production environment. While timelines can be somewhat streamlined by working with an experienced contract development and manufacturing organization (CDMO), given the challenges a developer may face along the way to scalable enzyme manufacturing, it is still crucial to gain an in-depth understanding of each process step and the obstacles they may still need to overcome. Quality from the start An experienced CDMO will want to determine what quality is required for a new project from the start.
It is crucial they have an advanced quality management system (QMS) to ensure that the right foundations are in place from the very beginning, including the utilization of fully qualified raw materials from trusted suppliers. Regardless of the desired output, it will be necessary to build in quality materials from R&D that are transferable to operations. Material selection must align to the latest guidance from local regulators.
In Europe, for example, materials used should be compliant with Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) regulations to progress to product manufacture. Sourcing particular GMP-grade materials can be a challenge, and sufficient time needs to be included in the project timeline to achieve this and qualify them into the CDMO’s QMS.
Typically, process development starts with consideration for the recombinant protein, the clone itself, and the cell banks. In some cases, a developer already has all of these in hand, but in others some pieces are missing which might mean a construct needs better expression, or a master cell bank must be created. Characterization of the cell bank is important to ensure that cells maintain their genetic and phenotypic properties. When working with Escherichia coli (E. coli), a common enzyme expression system, it is critical that cell banks are bacteriophage-free, as the presence of phages could have a serious impact on manufacturing operations.
For clinical trials, GMP-grade materials have their own defined standards but — despite a high level of quality assurance — the GMP cell bank might not provide the level of expression anticipated. Every enzyme has its own unique set of properties that need to be understood, adding layers of complexity to any manufacturing process. It’s here that experience is useful to manipulate conditions to achieve a combination of high expression, yielding good quality and active protein.
Enzyme manufacturing challenges
There are multiple challenging aspects of enzyme manufacturing related to fermentation. In E. coli, high cell density is achieved by careful manipulation of temperature, pH, growth and induction conditions. Managing the right balance of substrates and minimizing the formation of growth-inhibitory metabolic waste products can also ensure the culture is in a healthy state to maximize cellular productivity. Oxygen is a critical substrate to consider, as the ability to sustain the high oxygen demands achievable at small scale might be a bottleneck for a large-scale fermentation process.
To increase yield, some developers can also push the fermentation envelope too far. Getting the right balance is important. If the fermentation is not harvested at the right time, the recombinant enzyme can start to degrade. Companies may not have gathered sufficient data to understand the most productive point to harvest the fermentation, where cellular productivity is highest.
Fermenter occupancy time also comes at a premium and energy and material costs need to be considered, hence shortening fermentation run times can help to reduce manufacturing costs and complexity.
Additionally, although the technology associated with online monitoring is rapidly advancing, some monitoring is still performed by operators, like taking samples to measure enzyme activity.
If cellular expression during the fermentation process outstrips the rate of protein folding, it can cause the formation of aggregated protein into inclusion bodies. The ability to solubilize and re-fold from such structures is a significant challenge for the manufacture of many enzymes. In these cases, the ability to achieve active, soluble expression during the fermentation process typically becomes the main goal.
The complicated structure of some enzymes may also call for very targeted expression approaches. For example, if the enzyme has disulfide bonds, it might require a signal peptide to direct expression to the E. coli periplasmic space to achieve correct folding.
During fermentation, there can also be issues where the recombinant protein is toxic to the host cells. For instance, DNA modification enzymes can trigger an irreversible stress response in cells, leading to significant morphological changes. Selective induction methods and fermentation strategies to decouple cellular growth from recombinant protein expression can be adopted, although even low levels of unintended or “leaky” expression can still sometimes cause issues. Toxicity may be circumvented by producing inactive pro-proteins in cells for activation in a subsequent step.
Another consideration is the use of antibiotics, which are employed during fermentation for several purposes. One of them is appropriate selection of the right clone, as the plasmid containing the recombinant gene will be designed with an antibiotic resistance marker. This also helps with retention of the plasmid during the seed stages and, often, final fermentation production stage. Selection of the right antibiotic needs to be considered from the start. In particular, beta lactam antibiotics require very strict control given their hypersensitivity and are best avoided in a GMP setting. It is also necessary to demonstrate antibiotic clearance from the final product to ensure quality and safety.
Process development challenges
Complications during tech transfer are not uncommon. Everything hinges around the target specifications, such as host cell DNA or protein, but developers’ analytics may not be robust enough. Developers often have limited data as a result, which might mean they have identified a problem but don’t fully understand it. Purity is a common example, where some seek to use a SDS-PAGE technique to estimate, but that does not reveal enough detail.
In this case, mass spectrometry might be needed to understand different product forms. Further, all analytical methods have matrix effects, so every different condition that an enzyme is in can influence the result. A CDMO may also be at the whims of its external service providers in some instances, such as handling specialist test requirements, and so this needs to be understood and suitably built into the timeline.
Chromatography steps are utilized to achieve the high levels of purity required, and there are many factors to consider. These include the amount of protein loaded onto a chromatography column, as well as binding and elution conditions to separate contaminants from product. There is a temptation to maximize for full recovery but the result can include a poorly understood contaminant that then needs to be characterized. Ultimately, some developers try to achieve everything in one chromatography column, which can be difficult and lead to a non-robust process with repeat product failures.
Processes that improve purity can be complex and each additional step in such a process is an opportunity to lose product. It is a necessary challenge to simplify as much as possible.
To maximize yield, while still being able to achieve high purity targets. Affinity tags can be a useful way to purify enzymes but that approach is not always straightforward and can still lead to purification issues. Further, the tag can potentially impact the end-use performance of the product unless removed, and so the construct may need to be engineered to introduce a suitable cleavage site to remove it.
Scaling up
Scale-up goes most smoothly when strting from a good pilot facility, with scaled down tools that are representative of commercial-scale equipment. That includes fermenters, chromatography systems, tangential flow systems and centrifuges, so the developer and manufacturer can truly understand all relevant parameters throughout the entire process. This is not always possible as certain centrifuges are a particular challenge.
Large-scale disc stack centrifuges for continuous separation can be difficult to model. Even how you harvest cells can impact the effectiveness of a purification strategy. For instance, cells are recovered as a slurry from disc stack centrifuges and if cell wash steps are introduced, that can lead to unwanted lysis. Typically, cell paste produced by the expression system is frozen down to -80°C, and so storage capacity needs to be considered.
With E. coli as the host, the recombinant enzyme is typically expressed intracellularly and following lysis developers need to consider the vast amount of debris that would need to be cleared at a large scale, as well as the extent of nucleic acid and other contaminants that are released. Charged flocculants can help facilitate clearance of such material. This can reduce the high costs associated with using membrane filtration to achieve the clarity needed before proceeding with more sophisticated purification techniques.
As developers look to scale up, they can sometimes forget about how much chromatography resin they will need for the manufacturing process. As a result, they end up with very large columns and must incorporate cycling. This means holding material so that you can process part of it in one chromatography step, clean that, and start again. A developer can end up needing multiple cycles to purify an entire batch. This adds additional time constraints that can lead to stability issues.
Grappling with degradation
Some proteins are more susceptible to degradation than others. If protein degradation is apparent during expression, the use of protease knockout strains can help reduce or prevent degradation of the recombinant protein during the fermentation. Lysis and chromatography conditions, such as pH, buffer selection and temperature control, can also be manipulated to minimize degradation. However, there may be limitations imposed by the stability of the protein. For example, particular pH conditions might influence solubility. Certain additives can also be included to help stabilize the enzyme, such as protease inhibitors, reducing agents and metal ions. But using them can have consequences as the presence of metal ions, for example, can affect chromatography steps.
As a process moves toward large-scale manufacturing, there are other threats to stability. If conditions are not optimal, some enzymes can begin to degrade right away. Developers sometimes overestimate how much active enzyme they can get from cells, leading to an unpleasant surprise that gets compounded if they also didn’t account for degradation due to the increase in the timing of steps that can occur during scale-up.
Stability of the final enzyme product is a challenge with many different facets. Producing an enzyme with an appropriate shelf life is key, which is highly dependent on formulation. At early stages of development, however, it can be very difficult to predict. Some clients impose additional risks to stability because they are considering processing at ambient temperatures to avoid process complexity and facilitate transfer to a CDMO.
Cooling and cold rooms would otherwise be required, and not all CDMOs can offer this capability at large scale. Elevated temperatures enhance reactions, and if proteases are present, this can be a fundamental problem that requires stepping back into development to really understand whether conditions are sufficiently stable.
Innovation is often perceived in terms of a new technology, platform or piece of equipment. In reality, any advance that improves how a product comes into fruition is valuable and process development offers many opportunities for this.
Enzyme production comes with a large number of variables that must be considered in the design of a manufacturing process, and design of experiments can be helpful to home in on the most critical ones. It is important to carefully think through and design purification strategies that are amenable to large scale.
Understanding an enzyme product’s characteristics and properties can help guide development of the most effective processes. Companies often benefit from guidance from a process development partner with an experienced R&D team with synergistic capabilities and a full understanding of manufacturing constraints.
