Separation Science

Crystallisation Separations
Biocatalysis Separations
SpheriTech Collaboration

Sepagen specialises in the application of separation technologies to obtain molecules in the life-sciences having an improved purity profile. In particular, we focus on removal of impurities that have a similar molecular structure, such as stereoisomers and process-related impurities. Our in-house expertise concerns crystallisation and biocatalysis. In addition, we have an association with SpheriTech who have expertise in chromatography separations, and can offer solutions for obtaining pure products through a combination of these technologies

Crystallisation Separations

Although many companies have established a solid-state capability with an understanding of crystal forms, their knowledge generally applies only to the properties of a single substance. To separate compounds and purify them effectively, requires a more specialised expertise that Sepagen can provide. When the compounds concerned are structurally similar, then the separation/purification can be particularly challenging; this is often the case with stereoisomers and with process related impurities derived from a chemical process.

A common but often unrecognised reason for difficulty in separating structurally similar compounds through crystallisation methods is the formation of a solid-solution between the compounds. Here, the impurity can occupy sites in the crystal lattice of the required product. In a crystallisation step this may be represented according to the diagram below. At Sepagen we have the expertise to identify, quantify and often overcome the issues associated with such solid-solution formation to provide a robust separation process.

Representation of equilibrium between crystal and liquor phases where two components A and B form a solid-solution.solid solution equilibrium

Sepagen is particularly adept at using crystallisation methods to obtain single-enantiomer chiral compounds. This includes methods based on thermodynamic control, such as the 'classical resolution' method where a chiral compound is separated by way of diastereoisomeric salts, and/or by kinetic control through entrainment of the crystals of a required isomer. In such studies, we do not settle for an empirical approach, but set out to understand the physical chemistry parameters affecting a given resolution system. This includes for example, the analysis of the system through its phase diagrams, such as depicted in the diagram below.

Example of a ternary phase diagram between two components (n- and p-salts) in a solvent.example ternary phase diagram

Sepagen's current offering in crystallisation covers the areas listed below.

Assisting the Screening for a New Separation Method

We help our clients in finding new separation or purification methods for their products and intermediates. This will involve understanding the mutual crystallisation behaviour between the compound and the impurities, and where possible to obtain several chances for achieving a good separation. So if the compound will form a salt, we can design a rational screen for achieving the separation, ensuring that the results correspond to the actual physical properties of the salts and not just an empirical observation.

'Repairing' an Ineffective Crystallisation Process

It is our experience that crystallisation methods found initially for separation often fail upon process scale-up. This is often because they had only worked in the first place due to good fortune. Here we ensure that a redeveloped process is based upon sound thermodynamic principles, so that reproducibility can become assured.

Integration of a Crystallisation Step Within an Overall Chemical Process

A chemical process may be considerably simplified when advantage is taken of crystallisation. For example, conditions of a reaction, in terms of solvent, concentration and temperature, can often be defined so that the desired product crystallises directly from the mixture. Sepagen has particular expertise in combining the reaction with isolation/purification by means of crystallisation.

Biocatalysis Separations

The biocatalysis capability of Sepagen derives from a long history and track record of its Founders in the application of enzymes in the pharmaceutical industry, and who have developed many processes to chiral intermediates at kg to tonne scale. These utilised both commercially available enzymes and bespoke biocatalysts developed through microbial screening, cloning and subsequent production by fermentation. Biocatalysis is also applicable to the production of complex molecules for use as versatile building blocks in drug discovery, and it is from these foundations that Sepagen can offer unparalleled expertise and assistance to those companies wishing to exploit the benefits of enzyme chemistry.

Sepagen's current offering in biocatalysis is by way of subcontracting to Dr Steve Taylor, who is one of the original founders of Sepagen, and covers the areas given in detail below. His enzyme business is at Enzagen (accessed by clicking the logo below).

Enzagen logo

Access to Third-Party Enzymes for Screening

This requires a detailed knowledge of the enzyme supply market, and in particular can assist in access to most enzymes that are used in biocatalysis, for example, lipases, proteases, acylases, ketoreductases, and nitrilases. Of particular importance is our knowledge of the IP landscape, and we can address issues such as security of supply, as well as simplicity of use and availability on a price per kilo basis.

Access to Bespoke Enzyme Screening Panels

We can assist companies with developing their own enzymes. Screening panels can be created together with the supply chain development to reach a required manufacturing scale. We exploit the latest approaches to enzyme development, utilising database mining and informatics, and through a synthetic biology approach can rapidly assemble a panel of 5-40 enzymes from diverse sources for screening for a deisired biotransformation. This can be done cost effectively and a basic panel can be prepared in a matter of weeks. Such a panel can include both characterised and novel enzymes, guided by gene sequence data. A typical formulation is a powder ready for direct use in screening. Our approaches utilise simple well-proven expression systems that minimise the biocatalyst development time, and fermentation processes can be rapidly defined using standard cheap media.

Biocatalyst and Biotransformation Development

Sepagen can manage the transition from an enzyme found from a screening panel to having a manufacturing process. Through partnerships, we can have biocatalysts fermented up to 150 L scale (allowing typically tonne-scale manufacture of a pharmaceutical intermediate), including the downstream processing required. Enzyme formulation can be tailored to suit requirements for storage and stability. For the biocatalysis, Sepagen has extensive experience in both aqueous- and solvent-based processes, and we can integrate other specialised separation techniques, such as crystallisatio to remove unwanted stereoisomers or other impurities and by-products. We access designed polymeric supports for the immobilisation of enzymes and whole cells through our association with SpheriTech (see below); this can provide enzymes with advantaged physical properties, facilitating removal and reuse, and to achieve high flow rates when used in a packed-column setting.

Specialist Custom Synthesis

Whilst there are many custom synthesis and chemistry service providers, there are relatively few who specialise in separation technologies in general, and biocatalysis in particular. With its capabilities, Sepagen can construct relatively complex molecules with multiple chiral centres. We can selectively manipulate natural products (or mixtures of them) under mild conditions in order to attain higher purity, or to obtain new derivatives with advantaged properties. Additionally, we can perform 'cascade' reactions where two or more enzymes are used together for consecutive reaction steps, ultimately yielding more efficient processes.

Consultancy and Technology Evaluation

Sepagen is happy to provide consultancy work in biocatalysis ranging from simple evaluation of specific enzyme technologies, to market studies, intellectual property assessment and larger projects such as building an internal biocatalysis technology platform.

SpheriTech Collaboration

Propelled by the technological synergy, Sepagen has formed an association with SpheriTech in order to promote our joint capabilities; and to develop processes that use an element of technology from both our companies. The combination of chromatographic purification with both crystallisation and biocatalytic processes is a unique offering.

The SpheriTech laboratory sited in Runcorn, UK houses an extensive range of analytical and preparative equipment and is set up to handle projects from millgram to kilogram scale. Its preparative HPLC is capable of handling column loads in excess of 20g per injection.

Don Wellings, the founder of SpheriTech has more than 30 years' industrial experience and is an internationally recognised authority on peptide synthesis, polymer synthesis and chromatography. He is author of several texts on peptide synthesis and author of 'A Practical Handbook of Preparative HPLC'. SpheriTech has a portfolio of intellectual property covering a diverse platform of technologies including peptide synthesis, DNA synthesis, polymer synthesis, biocatalysis, cell culture and chromatography, that links in well with the capabilities offered by Sepagen. SpheriTech's first patent describes a novel platform technology for the manufacture of unique spherical polymeric particles.

Click the logo below for more information on SpheriTech.

spheritech logo

For further information please contact us at: enquiries@sepagen.com
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Sepagen Limited, Registered Office: Dalton House, 60 Windsor Avenue, London SW19 2RR, UK;
Company Number 6577610.