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Please find some commonly asked questions regarding our Praesto® pre-activated NHS, Epoxy and CNBr resins.
We've tried to be comprehensive, but if you have a question for one of our dedicated specialists, please contact us.
I am looking for alternatives to the Sepharose® CNBr and NHS resin from GE Healthcare. Can you support?
Yes, we have several alternatives with a range of bead sizes, designed to be 'drop-in' replacements without any changes to buffer formulations for the GE Healthcare products. Contact us to discuss specific process details so that we can offer our expert advice.
What is the preferred chemistry to couple enzymes?
The epoxide is separated by a spacer chain from the support of the material, allowing the ligand to maintain conformation and preserving activity. The number of epoxide groups on agarose can also be controlled quite well and maintain a more uniform distribution through the bead than some other chemistries, enabling effective coupling in the pore structure.
We can tailor the coupling reaction towards a primary amine, hydroxyl or thiol group on the ligand, allowing more versatility over other coupling methods. The resulting linkage between the resin and ligand will be more chemical stable than the ligand itself, preserving life time and minimising ligand leakage.
Is there a specific procedure/protocol that you suggest using, in terms of pH, Buffer type, time when coupling thiol-ligands to the epoxy agarose resin?
For certain proteins with engineered cysteine residues on the terminus, a reducing agent may be required to be added to the protein prior to coupling in order to activate the thiol groups on the ligand. If you get low binding it may be worth considering.
We would recommend a phosphate buffer system with a pH in the region of 9.5 (0.2-0.3 M DSP and 1-4 mM TSP) with resin to buffer/protein ratio of 2:1. If the ligand is stable at temperature we would recommend a 2-4 hour coupling time with constant mixing at 35°C or 24 h at RT. If possible, take a small portion of the supernatant during mixing to see if there is any ligand still in suspension, and to see if the protein has been bound to the resin.
Do you have any information about the compatibility of your resin with solvents, such as, acetone, DMF, and THF?
Pure and IEX resins are compatible with these organic solvents. They tend to swell to different extents with the solvents, but when the solvents are displaced they go back to the original size.
Agarose is quite resilient to most conditions except with a pH of less than 2, and strong redox agents.
Can you offer any advice on coupling conditions for activated resins?
Coupling epoxy resins to primary alcohols typically would only happen at pH 10 or 11. At pH 9.5 we typically bind to lysine groups and find it more effective than the lower pHs such as 8. pH 7 would be recommended for thiol coupling via the epoxy chemistry.
In the past, we have seen that we can orientate enzymes and proteins which we have immobilized on the resin so that the desired functional group isn’t accessible. However, this is more dependent on the pH more than the coupling conditions, as different primary amines become more accessible for coupling than the type of immobilization chemistry.
What is the content of agarose?
The agarose gel contains 6% agarose for our Praesto® Activated and IEX resins. Praesto® Protein A resins utilize a 4% agarose gel.
What's the ideal Epoxy Resin to buffer/protein ratio?
If the buffer volume is to large then it takes longer for all the protein to react with the epoxide groups on the resin, it is better to add the minimum volume of buffer which provides adequate mixing of the slurry.
Typically for 10 ml of gel we would make the total volume including buffer and gel to 15 ml, we use small process vessels for this and then this is scalable across vessel sizes. For smaller scale synthesis’ for this scale if using a vial on a rocking plate or similar you may need to adjust the amount of buffer.
As the agarose gel is 94% water we would recommend prewashing the gel in coupling buffer to replace the water in the gel prior to use as well so you don’t dilute the buffer.
Do you undertake any custom projects?
Our team of world-class researchers, polymer chemists and application scientists are experts at developing novel, customized products that meet the diverse requirements and expectations of our customers.
Whether you need a customized particle size, functional group, porosity or ligand density, Purolite Life Sciences can help you.
Our small-scale data shows higher pressure than at large scale, why?
Large-scale experiments are more relevant, as they measure the pressure drop over the resin. In smaller scale experiments, pressure restrictions from tubing and valves contributes to additional pressure drop.
What are the coupling conditions for Praesto® Epoxy?
For single point attachment we would recommend pH 8.5 using disodium phosphate, for lysine pH 9.5 using disodium phosphate and trisodium phosphate.
These simple steps should be followed:
1. Wash the gel in coupling buffer in the tubes centrifugation
2. Decant excess coupling buffer after washing so 1 ml of resin in 1.4 ml total volume.
3. Add 9 mg proA (if around 50 mg/ml concentration this should be around 0.2 ml – the total volume of 1.6 ml gives a good slurry concentration)
4. Add 0.35 g sodium sulfate (anhydrous) – we would avoid ammonium sulfate as it will shift the pH.
5. Mix at 35°C for 3 hours
6. Wash and deactivate remaining epoxide groups.
The reaction should be complete by this time with good coupling efficiencies.
What is the lifetime of Praesto® Epoxy when stored in water?
We have completed stability testing on the epoxy resin and found the stability of the product at 2-8°C to be 8 weeks in water. After this time there can be bug growth in the resin.
What is the ligand capacity on Praesto® NHS & Epoxy?
With Praesto® Epoxy it is typically 8-12 µmol/ml of settled gel – we can increase this if we don’t add a spacer.
Praesto® NHS is 12-18umol/ml of settled gel.
For small ligands it is possible to utilize most of the functional groups with yields of around 95% + if the process is optimized.
What is the ligand capacity on Praesto® CNBr?
It is very difficult to measure the functional capacity of a CNBr activation.
Typically, we release the product after it can bind at least 95% rspa Protein A at 3 mg/ml loading. As yet, we haven't failed to bind 9 mg/ml loading.
What is the recommended maximum running pressure of the 90, 65 & 45 micron beads?
We have not seen a significant difference in rigidity between the different particle sizes (45µm / 65µm / 90µm).
In smaller columns (5 cm diameter and smaller) we typically recommend to pack at 4 bar and not exceed 3 bar during operation. Data from larger columns suggests an optimal compression factor of 1.1 – 1.2.
Are resins manufactured in a cGMP environment?
Resins are not required to be cGMP accredited but in manufacturing we follow GMP guidelines appropriate to the product and the application. All relevant national and international regulations are adhered to.
The resin has to be manufactured to a set protocol where all raw materials used in its production are documented, the production is defined and documented i.e. operators, quantities and operations undergone are all recorded.
The product needs to be defined against a specification and released as such – i.e. purity, concentration and other key performance characteristic which is required by the process/product, and GMP appropriate to the product and its application.
What certification of quality has Purolite® received?
We are certified to ISO 9001:2015 standards.
Is Purolite Life Science QMS certified?
Yes, our systems certified against global standard ISO9001:2015The ‘QMS’ offers us a framework for our business processes, focusing on consistently delivering a product and service that meets our customer requirements and enhancing their satisfaction. Scope of the management systems captures the design, manufacture and sale process ensuring stringent quality requirements are maintained. Purolite Ltd is also ISO14001 and ISO45001 certified.
Who is our accredited, certification body?
Purolite Ltd is certified via BSi, who in turn are accredited globally.
Does Purolite Life Science consider other Healthcare guidelines?
Our customers’ requirements and intended use of our product is key for understanding ‘other’ regulatory and quality requirements. Therefore, we take guidance from both Pharmaceutical and Healthcare industries like IPEC-PQG for Excipient manufactures and ICH guidelines established to harmonise a global approach to technical requirements in the pharmaceutical industry. These translate into statutory requirements under EU and FDA CFR21 legislation critical to customers intended market. Understanding these requirements allows us to offer a product and service fit for its intended use.
Are Praesto® Product manufactured under cGMP conditions?
Production is not cGMP certified. However we adopt Good Manufacturing Practices prescribed in IPEC-PQG guidelines, our products are not tested to pharmacopeia monographs but industry method of test.
Are products controlled through supporting literature?
Yes, all commercial products are supported through comprehensive technical documentation; including Specifications, Certificates of Analysis. These are documented in our RSF (Regulatory support File).
Do you send CoA's with shipments?
Yes, all of our products are shipped with COA’s, MSDS and other regulatory documentation ensuring export and import condition are achieved.
Can I request additional certificates; Origin statement?
Yes, nonstandard requests are processed through customer special instructions, defined during the Sales Order process.
Is there documented change control system?
Yes, part of our QMS Purolite operates a documented and controlled Management of Change. Customer notifications are issued for any major changes.
What is the Shelf Life of Praesto® products?
Generally our products are allocated 5 year shelf life, unless otherwise stipulated.