SACHEM Displacement Chromatography

Three commonly asked questions on displacement chromatography

If you are purifying Monoclonal antibodies for therapeutic use, you cannot afford to ignore displacement chromatography. Below are three commonly asked questions about displacement. 

1. Can displacement chromatography be used for the production scale purification of antibody (Is it an alternative to using Protein A)?

---Cation exchange displacement chromatography has been done on antibodies using our displacers. Displacement as alternative to Protein A work has been done by Lonza in Europe and a patent is filed. 

2. What is the merit & cost cutting compared with the Protein A?

The price of Protein A is one expense that is less when using displacement but also the elimination of charge variants and the ability to load at higher levels translate into an overall cost savings. One of the key benefits of displacement chromatography is that displacement helps putting light into the science of mAb production. By detecting charge variants previously unknown in a substance, you can then determine whether these trace components found in a lab-scale process have the potential to impact the efficacy of your therapeutic when produced commercially. There is a huge risk elimination factor associated with this knowledge.

3. What data and real-world examples are available for scientists for further research on displacement?

---The isolation and elimination of product related impurities has been published by Genentech.  (Genentech's recent papers: Isolation and Characterization of therapeutic antibody charge variants using displacement chromatography and Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats.

Simply put, if you are purifying Monoclonal antibodies for therapeutic use, you cannot afford to ignore displacement chromatography using SACHEM EXPELL™-SP  for protein purification or ISOLIS™-SP for trace component detection. It is the best approach.

You might also find the following post interesting: Three reasons why we believe in Displacement Chromatography

Protein Purification: Achieve 10x increase in throughput, high recovery >70% and purity >90% by adding displacement chromatography to the mAb toolkit [2]

In their recently published paper Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography, research scientists Taylor Zhang, Justin Bourret and Tony Cano state "The advent of quality by design (QbD) in the biotechnology industry has increased the need for the isolation and enrichment of specific variants for biotherapeutic products [1]." MAb variants may form during any part of the antibody manufacturing process and their identification and/or isolation is critical as any of them can potentially affect the efficacy and safety of the therapeutic, the authors point out and continue that although the isolation and analysis of specific variants is a common requirement and "essential for assessment of biophysical and biochemical properties such as in vitro (potency and binding) and in vivo (toxicity, pharmacokinetic, immunogenicity) activity,"[2] their characterization is challenging due to the fact that most charge variants exist only at trace levels. The scientists  were able to more efficiently isolate mAb charge variants and increase thoughput 10x by supplementing their mAb characterization toolkit with displacement chromatography, "a powerful but unterutilized tool in the process development and characterization of complex biological therapeutics" [2]. 

 

[1] Rathore, Winkle. Nat. Biotechnology. 27 (2009) 26. 

 [2] Zhang, Taylor et al. Journal of Chromatography A Volume 1218, Issue 31, 5 August 2011, Pages 5079-5086 

 

Heterogeneity in antibody production in the biological drug discovery

Heterogeneity in antibody production is a hot topic in the biological drug discovery business these days.  It has long been known that production of antibodies in cell culture can lead to variation in antibody structure. However, it has yet to be shown that these variations truly have an effect in antibody structure and function in immunological assay or in circulating plasma. In a series of publications, Amgen scientists have conclusively demonstrated that circulating human IgG2 antibodies isolated from myeloma patients and normal serum appear to be a naturally occurring feature of antibodies belonging to the human igG2 subclass. (Wypych et. al 2008). In particular, they report the effects of disulfide bond heterogeneity, which can presumably affect antigen binding affinity and avidity and possibly affect the immune function at an organismal level.  While binding activity assays of the human IgG2 monoclonal antibody did not appear to be effected, this was not the case in an accompanying study (Dillon et. al 2008). In this accompanying report, they noted that disulfide mediated heterogeneity was indeed able to affect structure and function, which was manifested in several cell-based assays.

While the group was able to partially purify the antibodies on cation exchange columns to pursue their functions, they noted that further studies were necessary to fully characterize these variations in antibody structure. However, purification of these isoforms can be somewhat daunting. This is where the SACHEM approach of purification of antibody variants using displacement chromatography to isolate and concentrate the charge variants can be very useful. In fact, this approach has been used to generate   quantities of these variant antibodies for pharmacokinetic studies in animals (Khawli et. al. 2010). So for antibody groups who are looking for ways to conclusively nail antibody heterogeneity issues, displacement offers a unique and robust approach to solving these problems.

Resolution of Substances - How Two Methods - Displacement and Elution Mode Compare

Last week, we compared how substances emerge in the two chromatographic modes: displacement (as trapezoidal bands-the displacement train) and elution (as isolated, solvent-separated peaks). Today, let's have a look at the resolution of substances

1. How substances emerge
2. Resolution of substances
3. Required materials
4. Separating structurally similar biomolecules
5. Loading
6. Calculating isotherms

Resolution of Substances: Elution

In elution chromatography, for two substances to travel at different speeds and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. In order to achieve baseline separation of the peaks, operational simplicity and throughput are often sacrifices resulting in gradient elution and low column loadings.

 

 

Resolution of Substances: Displacement

The same interaction described for elution chromatography is also important to displacement. There is competition between the separating species for the sames site. The molecules that have the strongest attraction to the matrix bind and push the weaker species off to bind at a different site. This leads to sharpened adjaceing bands and better resolution int he displacement train.

The separation may be run at high loadings since there is no need to try to prevent component overlap. 

 

Three reasons why you should believe in displacement for preparative chromatography

 

We’ve devoted a lot of our recent posts to the application of displacement chromatography (DC) for analytical chromatography. Today, let’s talk a little bit about DC and preparative chromatography.

In the preparative mode your goal is to purify a product.  Why should you consider displacement chromatography for preparative chromatography? If you want to purify a product by chromatography and if you choose displacement you can get the following three benefits:

• Very high loadings
• High yield and recovery
• High purity

ALL AT ONCE (supporting data)

Why does this matter? Of the main methods of purification, chromatography is relatively expensive. If the chromatographic purification step is such a high value process, it will most likely be applied to purify high-value products (i.e. drugs – they tend to be expensive and need high purity). Given the benefits mentioned above, if you generally obtain a 30%  yield by other purification chromatography, with DC your potential yield could be around 90%. Doesn’t that sound great?

So, what is the Catch 22, you might ask. Overall displacement chromatography faces three challenges.

First, displacement chromatography is rarely taught in college programs. Most scientists are trained in elution chromatography and may not believe in the big promise that displacement chromatography makes. Furthermore and by no means surprising, many scientists quite naturally approach displacement chromatography with the concepts of elution in mind. However, displacement is set up in a fundamentally different way and one almost has to approach this process with a fresh mind. If you want to try out DC for your processes you will be most successful if you approach this process with an open mind, intellectual curiosity and willingness to "think outside the box".   Probably the biggest hurdle for successfully running DC for your purification process is you.

I have recently watched a movie called ‘The Code’ with Morgan Freeman and Antonio Banderas. In the movie, the protagonists Gabriel and Ripley are planning a big heist, the objects of desire being two prized Faberge eggs. The treasure is in a high-tech vault in a high-security building. The vault is secured with a 9-digit code – in essence, the protagonists of the movie not only have to get into the building but also figure out all the variables of the 9-digit access code to access their treasure.  While we don’t want to bring out the criminal mind in you and get you ready to plan a heist of your own, displacement chromatography brings up a similar set of challenges but also potential "treasures".

You have to "invest" into learning and optimizing DC for you. However, once you have the system set up, you are ready to speed along and reap the rewards and glory (minus the legal repercussions…;-)).

So, if you are one of those uncompromising, daring people that want to eat the cake and have it too…DC is for you. ;-) (P.S. Genentech and Lonza are among the "pioneers" trying our displacement chromatography for their processes).