HPLC is almost always injurious to proteins, pressures too high; GPC (gel permeation chromatography) is lower pressure but loses the fab advantages of regular HPLC (speed, tight peaks).

You’ll have to follow the US Federal regulations (CFR) for manufacturing a pharmaceutical. In brief, anything that ever touches the compound or is used to formulate it must be assessed, assayed and kept on record. Even for preclinical data you need to validate the equipment before any process. cGMP (current good manufacturing practice) procedures are detailed in CFR Title 21 Part 110 (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=110), and Part 211 (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=211)details production and manufacturing controls; knowing the rules thoroughly before making any purchases is wise. If the system you buy can’t be validated, it won’t be acceptable. For instance, ultrafiltration and column chromatography have their own special considerations; and computerized systems are another hurdle (21 CFR Part 11).

The point is, you can’t just clean up a product and sterilize it at the end: the entire process needs to be GMP. When you’ve narrowed your preference of a good method of purification, ask the equipment vendor if any pharmaceutical companies use that piece of equipment for manufacturing a like product. Do what those who have already succeeded before the FDA do-- your approval speeds through if there is a predicate compound already approved that was made the same way.

The ÄKTAready manual states it fills several regulatory standards including some FDA, and “the ÄKTAready system is biocompatible and hygienic, and meets all GLP and cGMP demands for Phase I-III in drug development and final-scale production.” Coming out of the people who gave us Pharmacia of course promotes confidence. Buying any machine designed with the CFR in mind should mean fewer potential pitfalls for you, so you can develop a shortlist between vendor assurances and the equipment used in other successful approvals. Your regulatory manager or consultant is a good partner in making this choice.

You’re right about the scale-up issue. Using a large-volume protein derived from nature, we used a separation process and proprietary industrial GPC we were able to sterilize and validate every step of the way, never had to switch. But if you don’t need large amounts for trials, the smaller scale may be more economical in the long run—the full-bore system is such a headache and time-intensive to validate all the time for small amounts. And if your compound fails trials…?

Any tannin considerations? A trial period for the system before purchase might be astute.

The primary factors determining a preferred separation method depend on parameters of size,ionic charge, solubility and density.High-performance liquid chromatography commonly known as HPLC is a popular protein purification technique due to the fact that it requires small sample and has high yield.

For native proteins/enzymes Akta purifier (old FPLC)

I agree! Akta purifier is the best one, in my opinion.

I am not sure that ACTA will be enough regarding capacity, for pre-clinical trial you will need more than1 g of your pure protein, anyway you can run ACTA several times.

For FDA you will want maximal reproducibility. So, if your protein can handle the higher pressure (many smaller proteins can), HPLC with a preparative scale column would be best. Otherwise, your next best option will be the FPLC. Both can be fully automated, and so are suitable for FDA, but HPLC usually offers more reproducible retention times so pooling the sample collected from full width at half maximum of the peak should give you a nice pure sample. I was able to purify > mg quantities of protein on the analytical scale HPLC columns during only one run, so g quantities are possible using preparative scale columns. Good luck!

HPLC is almost always injurious to proteins, pressures too high; GPC (gel permeation chromatography) is lower pressure but loses the fab advantages of regular HPLC (speed, tight peaks).

You’ll have to follow the US Federal regulations (CFR) for manufacturing a pharmaceutical. In brief, anything that ever touches the compound or is used to formulate it must be assessed, assayed and kept on record. Even for preclinical data you need to validate the equipment before any process. cGMP (current good manufacturing practice) procedures are detailed in CFR Title 21 Part 110 (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=110), and Part 211 (http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=211)details production and manufacturing controls; knowing the rules thoroughly before making any purchases is wise. If the system you buy can’t be validated, it won’t be acceptable. For instance, ultrafiltration and column chromatography have their own special considerations; and computerized systems are another hurdle (21 CFR Part 11).

The point is, you can’t just clean up a product and sterilize it at the end: the entire process needs to be GMP. When you’ve narrowed your preference of a good method of purification, ask the equipment vendor if any pharmaceutical companies use that piece of equipment for manufacturing a like product. Do what those who have already succeeded before the FDA do-- your approval speeds through if there is a predicate compound already approved that was made the same way.

The ÄKTAready manual states it fills several regulatory standards including some FDA, and “the ÄKTAready system is biocompatible and hygienic, and meets all GLP and cGMP demands for Phase I-III in drug development and final-scale production.” Coming out of the people who gave us Pharmacia of course promotes confidence. Buying any machine designed with the CFR in mind should mean fewer potential pitfalls for you, so you can develop a shortlist between vendor assurances and the equipment used in other successful approvals. Your regulatory manager or consultant is a good partner in making this choice.

You’re right about the scale-up issue. Using a large-volume protein derived from nature, we used a separation process and proprietary industrial GPC we were able to sterilize and validate every step of the way, never had to switch. But if you don’t need large amounts for trials, the smaller scale may be more economical in the long run—the full-bore system is such a headache and time-intensive to validate all the time for small amounts. And if your compound fails trials…?

Any tannin considerations? A trial period for the system before purchase might be astute.

From Dr. Atkinson's astute answer, and, for a small company, I would contract manufacture with an already cGMP facility (Boston Scientific?). We used Centocor for a cGMP manufacture of a diagnostic protein a few years ago. Always contract manufacture in the beginning, it is way less expensive than a personnel hire, purchase of equipment, and documentation necessary. Your CEO and CFO should know this!

The employer I mentioned wasn't Boston Scientific (which manufactures medical devices, not pharma). The earlier experience was back in the pleistocene when FDA needed to develop ways to approve and standardize proteins; several of the tests we decided showed health and purity of the molecule (such as isoelectric point and thin layer chromatography) were adopted.

But the Centocor idea is right on the money: an FDA-savvy contractor can keep you from reinventing the wheel, with all its mechanical breakdowns.

You would instrumentation that is compatible with proteins and won't denature the protein. Plus you would need that instrument to be approved by FDA for GMP, GLP guidelines. I would recommend the AKTA FPLC, both analytic and semiprep columns are available depending on the properties of the protein to be purified.

HPLC system for protein purification

Dr Atkinson's answer is very interesting, really qualified and pragmatic (not a common talent, I think).

anyway, in case Dr Zwiegelaar would already considered those aspects and decided for home-made production and purification, the FPLC systems -in my opinion- are enough to scale-up to grams (some) of proteins (Akta Explorer, in my hands).

An aspect that should be also evaluated is: to how many different proteins a single instrument will be dedicated, and the real needed amounts. Also the Xpress system could be considered (single workstation that controls from one to twelve purification modules (=twelve proteins at the same time), that perform two purification steps)

ps each module can be bought separately

Thanks guys, answers so far is very helpful. We will definitely be outsourcing our final manufacturing for clinical trials, so no argument there and thanks for confirming our decision in this regard. We would however prefer to develop the method in house and have a process available that can exactly be transferred and scaled up by the contract manufacturer with minimal process development on their side.

Thanks Kristine for the reference to the CFR articles, very helpful. We will have a look at what others are using. Was hoping to get a few "others" here that will be willing to comment on what they used to get their product through clinical trials.

Currently our choice would be between Akta Avant, the Akta fplc and Akta purifier. I am positive that the fplc or purifier would be able to do the job in the lab, but not sure about cGMP and compliance with all the regulations. Also the avant seems to be more capable of handling the upscale process than the fplc. Budget-wise we would prefer the fplc or purifier but only if it will comply with all the regulations.

GE is the right choice. We've been purifying proteins for some time now and we have come to the conclusion that unless you want to fragment a protein and detect fluorescent derivatives, GE equipment and media are impossible to beat. Sounds like they paid me, but its actually true. We have worked with BioRad and Waters systems and then come to this conclusion. Akta Explorer or Avant would be excellent choices. We even have an Akta Prime Plus, and even that works great, except that the low pressure limits dont allow tricorn columns that can be used with explorer or avant.

Thanks Samir, this puts me on the right track. I think I will repost my question under a new heading specifically asking people to compare the advantage of Avant over the less expensive fplc.

As for the column media used to purify the proteins, I have found the POROS media (see link below) to be a huge improvement over the old/regular FPLC media; low back pressure, hi flow rates, great separation, closer to HPLC performance. In my hands it cut down purification time 7 to 10-fold (including loading of sample and elution of protein). Also, 10-fold faster to clean-in-place the column/media for another run.

https://www.invitrogen.com/search/global/searchAction.action?query=poros&resultPage=1&resultsPerPage=15

Another system that is used mostly for smal molucule organic drug purification, MW=300-1000, is Biotage system, it is similar to AKTA but much easier to operate with a simple software, and that is more important for your case that is scalable up to 30L column and even large one.

Zoran,

Yes, POROS is up to 10-fold faster, especially with CIP. The max I can run my FPLC is only 8 mL/min and at that rate the back pressure was very low with POROS media during CIP, but the media can take much higher flow rates. When I used a 50 mL superloop to load samples in 6 M urea onto a Tricorn 5/50 column with the old media I had to load at 0.5-0.7 mL/min in order to not go over the pressure limit, I loaded the same column with POROS media at 5 mL/min and I was still well under the back pressure limit. Then when running a NaCL gradient in 6 M urea on the Tricorn 5/50 column, the old media had to run at < 1 mL/min, with POROS 5 mL/min was fine and was really too fast since my protein came off at a low NaCl concentration. Huge time saver.

Is there anybody here that have used the new Biorad NGC system? It looks impressive on paper but would like to hear from someone with experience using it.

1) I had worked at Down stream processing department (protein purification) in a bio-pharmaceutical company which manufactures biosimilars protein drugs. It has EU-GMP certified manufacturing facility and I was using AKTA systems from GE. If I have to use choose system for protein purification then AKTA would be my first preference. However, If you have doubt then you should select two of your favourite systems and talk with technical team of respective companies and let them know your concerns and questions. Believe me, technical team is the best resource to develop the right understanding about system and explore regulatory requirements related to the system.

2) I never had a chance to work with BIorad NGC, but one my friend did work with it and according to him AKTA sounds better system above all.

Cobus,

The other route to think about is purchasing a used system. The way the GE systems are made a used system should be as good as new, relatively speaking. And the cost savings would allow you to get the system you want, and still save money. I have bought equipment from Marshall Scientific at 1/3 the list price and it was like brand new, they have some Akta's listed (http://www.marshallscientific.com/SearchResults.asp?Search=akta+fplc&Submit=). Here is is another supplier (have no experience with them but they have lots of Akta systems): http://www.sci-support.com/categories/AKTA-fplc.html?gclid=CKWp37rzpLcCFU1p7AodgwIA_A. Lab-X is another potential source.

I used AKTA explorer and that was good for preclinical drug discovery studies on sickle cell disease tramsgenic mouse models.