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 

 

“Hidden” charge related monoclonal antibody (MAb) variants that can be isolated using Isolis™-SP

Charge-related heterogeneity is a common issue in nearly every monoclonal antibody process. It is clear that with the pharmaceutical industry shifting its focus to mAbs, there will be more and more types of heterogeneity uncovered.

A very nice review by Vlasak and Ionescu from Merck Laboratories was put together in 2008 and details quite a few of the currently well known charge variants and isolation by methods which exploit this charge variation (1). Briefly, while many of these variations resulted from modification at the COOH terminus, there were also conformational variants such as disulfide related variants, which, in a standard cation exchange chromatogram, would be very subtle and would run as a basic variant (2) on a cation exchange column. The acidic variants leading to conformational change may also show up due to loss of NH₂ through N-terminal glutamate cyclization (3).  While the main chromatographic approach to resolving or rather attempting to resolve these modifications is typically weak cation exchange chromatography, there are other lesser developed approaches such as hydrophobic interaction chromatography as well as reversed phase chromatography evaluating the antibody heavy and light chain fragments.  (4).

An advantage of reversed phase approaches is the ability to couple the analysis directly to mass spectrometry.  One common problem is that intact antibody heavy and light chains could possibly have these subtle variations masked in RP-MS analysis. Therefore, an additional step would be required to reduce the complexity such as enzymatic digestion prior to analysis or rather a proteomic approach.  In the sum of things, these types of analysis, while very sophisticated, often lead to diminishing returns as there is relatively little material to analyze further once the modified protein has been isolated and in the event of digestion for mass spectrometry analysis, it simply would not be possible to analyze for binding properties or pharmacokinetic properties.

Genentech has published a very elegant study, which shows that it is possible to isolate these diverse variants in enough quantity and efficiency to enable pharmacokinetic analysis of intact antibody variants (5). Their approach was to use SACHEM’s powerful cation exchange displacement reagents applied to their own chromatography resins. Not only were they able to separate various charge variants but they were also able to isolate them in quantity and significantly higher efficiency than using standard elution phase approaches. Vlasak and Ionescu list many variants that can be resolved and studied further using the SACHEM method. (Dr. Pat McAtee)

Contact the author of this article.

 

1. Vlasak, J. and R. Ionescu Curr Pharm Biotech. 2008, 9, 468-481.
2. Chu, GC. et. al. Pharm. Res. 2007,  24, 1145-1156.
3. Lyubarskaya, Y. et. al. Anal. Biochem. 2006, 348, 24-39.
4. Dillon, TM. et. al.  J. Chromatogr A 2006, 1120, 112-120.
5. Khawli, LA. MAbs. 2010 2, 613-24.

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. 

 

Mirror, mirror on the wall, who is the fastest-growing segment of them all?

Sales of biologics totaled $94 billion by 2007 and represented the fastest growing segment of the $600 billion pharmaceutical industry.[...]

• The total global market for protein-based therapeutics is projected to grow between 7% and 15% annually over the next several years.

Among the most prominent blockbusters are mAb-based products indicated for treating cancer; insulin and insulin analogs and EPO-based products.

(Source Nature Biotechnology Vol 28 Number 9 September 2010). Request a copy of the article.

 

 

 

What does this have to do with displacement chromatography? Read how Genentech utilized Displacement chromatography for mAb charge variant analysis to understand their processes and make them more effective.

 

Genentech article on displacement chromatography for mAb charge variant analysis

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants pose a challenge to demonstrating product comparability. Check out the report published in Landes Bioscience Journal "mAbs" (Vol 2 Issue 6) to read more on how Genetech  gained further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity by utilizing cation exchange  displacement chromatography to isolate the major charge forms of a recombinant humanized IgG1 and compare their in vitro properties and in vivo PK.

To access the full report (subscription based) and read how Genentech utilized displacement chromatography for mAb charge variant anlysis, please follow the link:

Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats

 

Need more reasons or supporting data to believe in displacement chromatography? Or do you want to join your fellow scientists and try out SACHEM's QuickStart™ Displacement Starter Kit™, an "all-inclusive" package that provides an introduction to displacement chromatography and opportunity to evaluate this tool for your processes?

 

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What does the recent FDA approval of a generic version of Lovenox® mean for the Future of Biosimilars?

Recently, the U.S. Food and Drug Administration granted marketing approval of the Abbreviated New Drug Application (ANDA) for enoxaparin sodium for injection, a product designated as “generic equivalent” to Lovenox®.  While the product is not protein-based, it is an extremely complicated molecule to make and refine - similar to attempting to reproduce a biologic.

What does the recent FDA approval of a generic version of Lovenox mean for the Future of Biosimilars?

The “Business of Biosmilars” interviewed and posted an insightful podcast with Mark McCamish, the Global Head of Biopharmaceutical Development for Sandoz International, a Division of Novartis. Sandoz is the world leader in development and commercialization of high-quality biosimilars.

The interview with Dr. McCamish touches on a few points that the DC Blog finds very interesting:

According to McCamish some of the key technical hurdles companies will have going into the biosimilar space are: “One really needs to have world class analytical characterization capabilities such as those actually evaluated by the FDA. One has to have the capacities of modifying the production process of the biosimilars to achieve a specific change in the molecule to make it closer to the originator molecule based on comparative data. Sometimes this takes multiple iteration, which requires specific skills, times, and resources."

Secondly, “global companies have to provide data on the comparability of the product to the original product.  If there are differences, a company needs to provide data that convinces the regulators that those differences do not alter the safety or the efficacy of the product. “

Since displacement chromatography simultaneously enriches minor components and separates those from major components, its a unique tool in these applications. 

To listen to Dr. McCamish’s expert industry perspective on recent developments, please follow the link to the podcast. 

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When a new technology runs...do you want to be part of the steamroller or part of the road?

The DC Blog came across this funny quote. (We don't know who is the originator and can only credit this to "unknown").

 

"When a new technology runs over you, you're not part of the steamroller, you're part of the road."

 

The quote resonates with us (aka the DC blog) because we all know, change is constant – it happens all the time. It’s hard to keep up with technology because life, business, research…well, everything moves at a fast pace and it’s hard to find huge blocks of time to really learn new skills. 

 

Compared to elution chromatography, displacement chromatography (DC) is a new technology. Granted, it has been “on the market” for a while but more in the form of a "sleeping beauty" - dormant - for the past 100 years. For one, displacement chromatography is not one of the prominent or popular subjects taught in biology or chemistry programs. Secondly, for a lot of applications elution chromatography does its job "just fine"  – so why change? Where's the steamroller?

 

When it all boils down, the vehicles that will get you from Point A to your destination are often plentiful. A motorcycle, a Porsche, a truck or a bicycle  - all of the above will get you to and from one place. It depends on how fast or slow you want or need to get somewhere, how many resources you have and what you are trying to sell...and to whom. Needless to say, the same applies to analytical tools and "vehicles" in biotech. New drug therapies and biotechnological innovations are coming to market at a fast pace. Whereas for some, current technologies work great, others are already calling for new or additional tools... which brings us back to displacement chromatography.

 

When trying out displacement chromatography for the first time, the DC "pioneers" are faced with a multitude of questions. We have received questions about the displacer itself, about which column to use, which buffer?  How should you clean the column?  How do I run the experiment?  What are the variables in the experiment and how do I adjust them right? How do I know if I am successful? 

 

Given the many time constraints we all face, we want to help you stay on top of the new technology steamroller, so we’ve come up with the first of what will be a series of displacement purification Quick Start Kits.  The kits are all inclusive---they include a pre-screened column, the charging solutions, the buffers, the displacer, the cleaning agent and in the case of our first kit, a standard.  With the kits, we supply the protocol, the displacement “train” and give you the step by step guide to walk you through your first optimization of DC.  The column is reusable and you can order a refill kit of displacer, buffers and cleaning agents anytime.  The goal of the kit is for you to quickly learn how the parameters of DC and how you can achieve better results for your process so that hopefully we are making your life just a little bit easier.  Ready to try it out?

 

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Bioanalytical Tools to Demonstrate Sameness in Biosimilars

According to Genetic Engineering & Biotechnology News article "Complex Biosimilars Demand New Rules" by Thomas B. Smith, M.D., more than $10 billion worth of biologics are expected to be off-patent by 2010, creating enormous incentives for the development of generic alternatives to these drugs. As a result, it is anticipated that the market for biosimilars will grow significantly in the coming years.

Smith goes on stating that complex, protein-like drugs and biologics offer unique challenges to both drug-development professionals as well as regulatory decision makers. The FDA’s conclusion (in 2006) regarding these protein-like products noted that the complexity of these products in effect made them “unknowns” for which currently available chemical and bioanalytical tools likely are not able to demonstrate sameness.

The DC Blog wants to point out that we think that Displacement Chromatography has the potential to be such a bioanalytical tool able to demonstrate "sameness" in biosimilars because:

• DC lets you find charge variants that you can't find normally
• DC has the potential to reduce high cost in your process
• Keep in mind, DC is not for everyone or for every process. It is like a rocket ship that - with preparation - can get you to the moon...but you might not need to get to the moon. If you want to give DC a shot, try it out with SACHEM's Quickstart Displacer Starter Kit.

Watch our 2 minute video to see how displacement chromatography works.

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