An up-close look at biocatalysis: Accelerating synthesis using nature’s catalysts

Watch our webinar on biocatalysis as Mark Muldowney, Head of Technology and Innovation, and Greg Holgate, KTP Associate, discuss the benefits of using nature’s catalysts to accelerate syntheses.

Hi, I’m Mark Muldowney and I’m Head of Technology and Innovation at Sterling Pharma Solutions, and I’m joined today by my colleague, Greg Holgate.

Thank you, Mark. My name is Greg Holgate, I’m a Research Chemist working here at Sterling in collaboration with Northumbria University, and currently I’m undertaking a 24-month EPSRC, an Innovate UK-funded knowledge transfer partnership (or KTP). I’ve been working on this now for approximately 18 months.

Thank you Mark and Greg. Today we are going to discuss biocatalysis, an area that has experienced tremendous growth in the pharmaceutical industry in recent years. Today we’ll be starting off by providing a general overview of biocatalysis. analysing its growth and highlighting its key benefits. Next, we’ll be discussing the immobilisation of enzymes and its advantages for the pharmaceutical industry. We will then shift the discussion to Sterling and our approach to biocatalysis. Finally, we will end with a case study to provide a greater insight into Sterling’s biocatalysis solutions. With that, I’d like to turn over to Greg.

Thanks, Charlotte. Let’s begin by discussing exactly what biocatalysis is and what’s driven its growth.

Since the 1970s, biocatalysis, or the use of enzymes to speed up chemical reactions, has gained significant traction in a wide variety of industries, including that of pharmaceutical development and manufacturing. Major technological advances have enabled more efficient enzyme discovery in manufacture, and this has led to an unprecedented growth in the demand for such technology, with the global market for biocatalysis anticipated the grow by approximately 15% between 2019 and 2024.

So, within today’s webinar, we’d like to discuss some of the key concepts behind the use of enzymes as catalysts, why we think it’s so advantageous, and how our approach to biocatalysis at Sterling delivers superior solutions to our customers.

To begin, it’s worth taking a look at the history of biocatalysis as we know it today. Records show that ambient yeasts were used in brewing and fermentation processes around 8,000 years ago. However, in these times there was little regard placed upon what was actually responsible for accelerating these highly demanded processes. It was only until 1833 that Chemists, that were working in a sugar factory in France, first isolated and purified the enzyme diastase from its ancillary cellular components. This was the first record of enzyme sulfurite extract being yielded. However, from here it took a further 60 years for the German Chemist, Edward Buchanan, to show that enzyme catalysis could be harnessed X-VIVO, or without the presence of a living microbe. Subsequently, thousands of novel enzymes would have been discovered for a multitude of different chemical transformations, allowing biocatalysis to reach mainstream status around 20 years ago, and since that time is continued to become more and more widespread across industries. Including food and beverage, cosmetics, waste treatment, and of course pharmaceutical development and manufacturing.

So, what scientific advances have paved the way for commonplace implementation of biocatalysis in manufacturing? Firstly, next generation methods in DNA sequencing have revolutionised genomic research. With ultra-high throughput capabilities, genetic material can now be deciphered rapidly compared to the conventional or old cyano methods of DNA sequencing. This has created tremendous amounts of data which can be harnessed by researchers in vast open access databases, such as TREMBL. Secondly, through directed evolution in protein engineering, Scientists have developed methods to tailor and optimise enzymes to suit the needs of a particular reaction via the manipulation of their DNA code. It was only in 2018 that the Nobel Prize in chemistry was awarded the American Chemical Engineer, Francis Arnold, for work in directed evolution. Finally, developments in enzyme immobilisation have yielded highly efficient robust and stable heterogeneous biocatalysis. This is directly resolved some of the key issues regarding the use of enzymes at industrial scale and we’ll talk about this in more detail in slides to come. From here I’d like to pass back over to Mark now who’s going to discuss some of the key advantages of biocatalysis.

Thank you, Greg. There are several key drivers for the recent growth in biocatalysis. A number of scientific breakthroughs have led to increased commercial usefulness, and we’ll now take a closer look at some of them.The first, and most obvious, advantage is efficiency. As its name suggests a biocatalyst by its very nature reduces the time needed for a chemical reaction. Although acceleration rates vary from enzyme to enzyme, examples like the phermophyllic carbonic anhydrase SARS CA, which converts carbon dioxide into bicarbonate, the form reactions are pretty much diffusion rate controlled.The next key advantage of biocatalysis is customisation. Natural catalysts have a very specific purpose. Through optimisation, scientists can tailor enzymes for a particular process. In this example, the HIV drug, Islatravir, was produced by a multi-step biocatalytic cascade. Several of the enzymes involved were the products of directed evolution, and this increased the overall yield from 15% to 51%.

Another example of the benefits of enzymes is in their cost effectiveness. Although many novel enzymes can be relatively expensive, under the right conditions they can be recycled, and Greg will talk more about that later. Also, due to the high chemo and regio selectivity, side reactions are limited, and costly protection and de-protection steps can be avoided.

And finally, biocatalysis is environmentally advantageous. Enzymes are made from renewable resources and most enzymatic reactions are performed in aqueous systems. Sustainability is particularly relevant for the modern pharmaceutical industry right now, and it aims to shift towards greener practices. At Sterling, developing sustainable processes is a key driver in supporting our customers in achieving their environmental goals. For example, using up earth supplies of heavy metals in chiral catalytic reactions will not always be acceptable.

Now I’d like to hand it back over to Greg who will discuss the benefits of immobilisation.

Thanks a lot, Mark. We’re going to take a look now at what enzyme immobilisation is and how exactly it has overcome some of the key challenges of using enzymes at scale. Before we take a look at the power of immobilisation and just what it is, it’s important we discuss the key challenges of using enzymes in synthetic chemistry.

Firstly, enzymes are very sensitive and fluctuating conditions of storage or use can denature to enzymes rendering them less efficient or ineffective altogether. Secondly, there’s a widespread concern, especially in the pharmaceutical industry, that enzymes may end up in end product and thus compromise its purity. This is unacceptable and working with biocatalysts requires a partner that has specific quality controls in place to avoid this from occurring. Thirdly, despite their ability to reduce process costs, enzymes come at a relatively high upfront expense and anything that can be utilised to offset this cost is a welcome advancement.

This brings me to immobilisation, which is the process by which an enzyme is either affixed to or encapsulated within a heterogeneous solid support. This can be done via variety of physical or chemical means including adsorption, covalent bonding, cross-linging etc. and immobilisation supports, or media as we often call them, are polymers or inorganic materials such as polyacrylates or silica.

In the forthcoming sides we will discuss how enzyme immobilisation directly addresses all three of the key challenges facing industrial biocatalysis as we spoke about on the previous slide. The stability of an enzyme to. conditions such as temperature, pH, and organic solvent content is significantly improved through immobilisation. This is because a stable micro environment is created for the enzyme to operate in when affixed to pause within the solid support. In the example shown, an immobilised alpha chymotrypsin is shown to be one thousand times more active in a methanol environment compared to its free enzyme counterpart. Enhancement and stability are significant as it allows greater flexibility in the conditions for enzyme use and the substrates with which they can act upon. Next, immobilisation aids in the process of enzyme separation from end products. Without immobilisation, enzymes can remain within the end product and are actually quite challenging to detect through typical analytical methods like HPLC. With immobilisation however, the enzyme is attached to an insoluble solid support, and this means that a simple filtration can completely remove any traces of enzyme from the end product. In the example shown an immobilised lipase called novazymphosate 435 was showing to produce a pure triglyceride over 15 cycles of filtration and reuse, without any diminishment of enzyme activity. This suggests that little new enzyme had been leached from the immobilisation support and actually all had been retained within the heterogeneous catalyst. Immobilisation therefore should provide full confidence that end product are. of the quality standard required for pharmaceutical manufacturing.

And finally, immobilisation represents a significant cost reduction as it enables enzymes to be separated, stored and, most importantly, recycled. This is particularly appealing for the pharmaceutical industry because it represents a direct cost saving in the manufacturing process which is very compelling when you’re dealing with large quantities of what is typically a highly expensive material. The number of times a biocatalyst can be reused varies but in one example of a process used on site here at Sterling, an immobilised lipase has been recycled and reused for more than 20 batches over several campaigns. This is essentially diminished the upfront costs of that enzyme by about 20 -old, which is really quite remarkable.

I’d like to turn it back over to Mark now, where he’s going to discuss some of the things that differentiate our approach to biocatalysis here at Sterling.

Thank you. The content we’ve covered up this point in the webinar are centred on general biocatalysis. I’d like to now focus on the approach we take at Sterling to be our customers choice of biocatalysis partner.

We believe that many of our strengths in other fields make Sterling a compelling partner for biocatalysis projects as well.

First, at Sterling we are experts in complex and hazardous chemistry. This has been proven over our 50-year track record. The care required to handle hazardous materials safely, for example with accurate temperature control, is also beneficial for handling temperature sensitive biocatalysts. Next, we support our customers programs from discovery through to commercialisation. As skilled API developers and GMP contract manufacturers, we have been utilising enzymes for many years now, for our API customers.

Thirdly, we are partnership development and manufacturing organisation, or PDMO^®. That means we hold ourselves to a high standard for collaboration, communication and transparency. We bring this PDMO^® model to each project and this is vital, especially when scientific complexity makes transparency even more critical.

And finally, Sterling continues to be committed to innovation in pharmaceutical API development and API manufacturing. To advance its commitment, we work closely with leading academic institutions and technology providers who are widely known and respected for their expertise in biocatalysis.

For example, a range of partners not only assist dealing in the initial screening and optimisation of enzymes, but also in the scale up and commercialisation activities.

I would like to now mention a few of our academic collaborators. Firstly, Northumbria University is world renowned for their expertise in biocatalysis. Through Greg and the KTP program, we are working with Northumbria’s nzomic division on customisation of enzymes through directed evolutions, expression and immobilisation.

The second key academic partnership is the University of Leeds. We work closely with their EPSRC Centre for Doctoral Training on novel techniques of immobilisation. Their expertise will play a pivotal role in allowing us to employ immobilisation for the benefit of our customers commercial manufacturing.

We are confident that this network of partners, together with our proven expertise and PDMO^® approach, will deliver our customers best-in-class biocatalysis solutions that raise a bar for innovation and quality. Now, there are several additional advantages to partnering with Sterling on your next biocatalysis project. For example, IP ownership of your enzyme. Anyone working in the pharmaceutical space knows the importance of this one. You’re investing in the development of a biocatalyst for your compound, so here at Sterling we believe that you should own the IP. Also we provide the potential to store and recycle enzymes. This is aligned with our commitment to sustainability and optimisation process efficiencies.

In addition to having access to our expertise, our customers also gain access to the academic and scientific partners we’ve just discussed. The science behind industries use of biocatalysis continues to advance and evolve, so I can’t overstate the value of this particular advantage to our customers.

As well as the ability to scale up enzyme development and biocatalysis reactions, through the capabilities of Sterling and our partnership network, by working with Sterling on their biocatalysis projects our customers also gain access to full cGMP manufacture of APIs and intermediates, our core skill set. I’d now like to pass you over to Greg who’s going to go through one of our Sterling case studies.

Thank you, Mark. To finish off our webinar I’m going to talk about a current biocatalysis project that we’re working on here at Sterling, wherein we’re investigating a dual enzyme cascade reaction of a highly substituted benzoic ester.

Utilising the augmented panel of enzymes that we have available on site, as well as our close biologic suppliers, over 50 enzymes were screened for the two-step conversion. This allowed us to identify some weak hits which were then screened against 22 structural analogues, which were either prepared by chemists in-house or purchased from suppliers. This allowed us to undertake what we call a retro structure activity relationship study, which allowed us to identify some more enzymes from the literature with activity against compounds of high structural similarity. These additional enzymes were then essentially investigated and subsequently expressed, which allowed us to identify some more hits which were highly efficacious for the two-step conversion. From the structure activity relationship study, we were also able to paint a picture of some of the key interactions our compound was having in each enzyme active site. To solidify our findings, we’re currently modelling the enzymes in silico for crystal structure characterisation and active site resolution, and we anticipate that the data that we generate from this will allow us to optimise the enzymes via the root of directed evolution or protein engineering should it be necessary.

Whilst we’re investigating the conventional route to the customer compound, we acquired data that suggested an alternative biocatalytic route may be feasible. So in exploring this route, a highly efficient enzyme for the conversion of the benzoic ester was found. Though the enzyme was supplied in its cell free extract form. The free enzyme was therefore screened against 15 distinct types of immobilisation media, with an array of different affixation techniques including ionic bonding, absorption, covalent and so on. From these studies, significant enzyme activity has been retained upon many of the immobilisation media tested. With next steps including optimisation and recycle studies. In the future, this process may be examined for the continuous flow synthesis of the customer compound.

I’d like to pass back over to Charlotte now who’s going to conclude the webinar.

Today’s webinar has provided the general overview of biocatalysis, its growth in recent years, and has highlighted its key benefits. We have then discussed the advantages of enzyme immobilisation for the pharmaceutical industry. We have discussed Sterling’s approach to biocatalysis and reviewed a case study to provide greater insights into Sterling’s biocatalysis solutions. Thank you for joining today’s webinar.