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

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.

What is your epigenetic destiny? Can you identify and control it?

Identical twins have the same DNA, the exact same genes and the same DNA. Two people start out the same, but their appearance and their style can change the way they look. Our lifestyles and environment can change the way our genes are expressed, leading even identical twins to become distinct as they age.  

Imagine this: one identical twin gets diagnosed with cancer, the other is perfectly healthy. Or here is another example, how can one twin develop a metabolic disorder whereas the other stays "in shape"? How can two genetically alike individuals be so similar and so different?

Some researchers have discovered a new bit of biology that can work with our genes or against it: Epigenetics. Epigenetics literally translates as “above the genome" [i.e. the epigenome is the control center or HQ of a cell - in very simple, technically not 100% correct terms]. Identical twins can be genetically identical, but may not share the same epigentical destiny, especially if their lifestyles differ. The epigenome acts similar to the software in a computer telling a cell how it should act; an epigone can turn on or off a certain genetic function. (If you want to learn more, watch this great introductory video by NOVA Science here.)

Epigenetics is a subtle modification that affects metabolism, malignancy, cognitive function, and even itchy, scratchy feet!

It is possible to search and identify the epigenome using two dimensional displacement chromatography on an analytical scale. (Displacement chromatography enables you to separate very closely related components that have very subtle differences.)

Once identified, compounds [and eventually drug therapies ] that target certain epigenetic functions can be developed, i.e. to control cancer or obesity cell function. This represents a significant step towards characterizing the epigenome with respect to the variation in the non-inherited phenotype.

 

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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Low Abundance Biomolecule Detection - Article in GEN

Genetic Engineering & Biotechnology News published an article: "Low Abundance Biomolecule Detection" in their July edition (Vol. 30, Nr. 13).

The article features a case study by CMC Biotech Working Group on the development of a monoclonal antibody that shows the favorable outcomes possible with displacement chromatography. 

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Trends in Biopharmaceutical Development - Biosimilars

Howard Levine of The BioProcess Blog recently wrote "The future of biosimilars," a great overview of the regulatory landscape affecting the biosimilars market. Levine points out that the momentum and interest in the biologics market is growing, however, that the complexities of developing and manufacturing biologics can make the market entry for "newcomers" daunting. Guidelines and regulations for the approval and marketing of biosimilars already exist in Europe. According to Levine, pending biosimilar legislation in the U.S. is currently more favorable to innovator companies than to those developing biosimilars. Current proposals for biosimilars legislation call for extensive clinical trials and do not grant substitutability of the biosimilar. This means that the biosimilar product will have to be specifically prescribed by the physician and pharmacies cannot freely subsitute a prescription with a biosimilar thus requiring heavy marketing of the biosimilar alternative in addition to the clinical trial expenditures.

Levine points out that companies developing follow-on/bio-better or biosimilar products will most likely use the traditional "innovative medicine" route in the U.S.. Levine uses TEVA's Neutroval/BLA for XM002, a biosimilar of Amgen's Neupogen. as an example. The product has been approved as biosimilar "TevaGrastim" in the EU but was filed as a new medicine and not a biosimilar in the U.S. (read the whole blog post here).

As any market cycle goes, traditional blockbuster drugs might soon have competition from biosimilars, especially with the market drivers of high population growth, lifestyle disease growth and the global economic development. While legislation for biosimilars in the U.S. is lagging, there are already guidelines and regulations in parts of Europe and Asia (The Department of Health (DOH) in Taiwan recently approved the first drug permit license to a biosimilar which shows that Taiwan government has the expertise and capability to review biosimilar drugs.)

 

 

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Orthogonal Chromatography, Displacement and Proteomics- Custom Separation of Biomolecules

Resolution and substance! As in DaVinci’s drawing of polyhedra in De Divina Proportione,  multidimensional chromatography is predicated on the concept of orthogonal relationships.  Or as George Clinton in Parliament might have put it, “chromatography may solve but orthogonal chromatography will resolve”.

Orthogonal can mean many things but in chromatography, it loosely refers to a complimentary but different form of purification from the initial purification, that is orthogonal chromatographic systems provide different information about a substance.

For instance, it is common to proceed from ion exchange chromatography to gel filtration chromatography as the two techniques are complementary and compatible in a direct sequence in either direction. Plus, the investigator gets the advantage of separation by charge in one run coupled with the separation by size in the other run.  The same relationship exists between anion or cation exchange chromatography and hydrophobic interaction chromatography.

With the advent of user friendly mass spectrometry, it has become possible to run reversed phase liquid chromatography  and mass spectrometry in an on-line sequential manner.  As various genomes have been sequenced and catalogued, proteomics has become highly popular. LC-MS systems are now commonplace in analytical laboratories, both in academic and industrial settings.  Typically, a protein sample is digested with an endoprotease such as trypsin , then “desalted” and loaded onto a reversed phase chromatography column. The post column flow is split between a detector, such as uV or an electrospray assembly coupled directly to a mass spectrometer. From the mass spectral information, the amino acid sequence as well as posttranslational modifications is readily detected. 

Orthogonality has come from on-line two dimensional chromatography in which the peptide mixture is first run through a cation exchange column.  The eluted peaks are then run directly onto a reversed phase column couple to a mass spectrometer. It is possible to create a capillary tip where cation exchange and reversed phase resin are adjacent to one another. However, this approach has limitations when run on-line due to the inability to switch between resins readily during development of the gradient in the first dimension as well as the generation of huge amounts of data to be analyzed. This hurdle has been overcome by using step gradients in the first dimension followed by linear gradients in the second dimension.

Still are investigators getting the full picture and the resolution they crave?

 

Previously accepted two dimensional protocols result in analyte yields which are typically very low, requiring analytical sensitivities in the picomolar to femtomolar range. Displacement chromatography offers the distinct advantage of concentration of trace components. Two-dimensional chromatography utilizing displacement (click here for instructional video) rather than elution mode in the upstream chromatography step represents a potentially powerful tool for analysis of trace components, modifications, and identification of minor expressed components of the proteome.

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Find the Needle in the Haystack

Displacement Chromatography is well-suited for isolating and resolving minor species from one another. In displacement mode, components are forced to compete with each other for binding sites, exploiting small differences between the components and forcing them to self-segregate as they move down the column.  Below is an example of how this chemistry could be advantageous in applications such as the deorphaning of G protein-coupled receptors (GPCR). By using displacement mode, one is able to isolate as well as concentrate trace components with excellent recovery:

Nowadays every biomedical scientist is familiar with the 7 transmembrane receptor class, the G protein-coupled receptors (GPCRs), which have taken center stage in drug discovery and the study of cellular growth and differentiation. Almost exactly 20 years have passed since the first human GPCR was cloned and characterized by Jeremy Nathans while he was a graduate student at Stanford Medical School. Another two years passed before a team from Merck Research Laboratories, Duke University Medical School, and Howard Hughes Medical Institute, headed by Richard Dixon and Robert Lefkowitz, cloned the ß2-adrenergic receptor and showed that it was homologous to rhodopsin. A frenzy of receptor-gene cloning followed, leading to a new era of molecular pharmacology based on some knowledge of receptor structure. Taxonomies of hundreds of cloned receptor genes suggested evolutionary relationships among receptors for widely disparate natural ligands: from proteins, to lipids, to nucleotides, to calcium. Soup to nuts, GPCRs could apparently feast on almost anything.

Despite the cloning frenzy of the 1990s, few were prepared for the vast number of so-called orphan receptors identified by the Human Genome Project (Nice intro/overview on YouTube). Orphan receptors – those genes that have the molecular hallmarks of GPCRs, and are expressed, but have no known endogenous ligand – became targets for extensive "deorphaning" programs, mainly in the maturing biotech industry. The true orphans were those receptors without known endogenous ligands or small-molecule pharmacophores. The hope was that many of those receptors might be drug targets for the next generation of block-buster therapeutic agents with billion-dollar-per-year sales. After all, small-molecule GPCR modulators have been estimated to make up more than one-half of all commercial pharmaceuticals, including some former best-sellers such as the ß-adrenergic receptor blockers, and more recently the selective serotonin reuptake inhibitors.

Deorphaning a GPCR is not exactly a walk in the park. One starts with a whole organ or two and through multiple fractionations and purifications-losing precious material all the way if it is there-hopefully ends up with something that will light up the receptor in assay usually done on a device called a “FLIPR” or a Fluorometric Imaging Plate Reader using cell lines that have been made to express the orphaned GPCR.

While the downstream aspects of this work are pretty much assembly line and cut and dried, the isolation of the unknown ligand is a brute force adventure in purification torture as the ligand is a minor, transiently expressed product.  Traditional HPLC separation techniques are really not well-suited for isolating trace components; larger columns do not provide the necessary resolution and analytical columns require multiple cycles and significant sample handling, which can further erode recovery of something that is present in trivial amounts. 

Displacement Chromatography is well-suited for isolating and resolving these minor species from one another. Watch how DC mode forces the components to compete with each other for binding sites and how displacement mode provides an opportunity to isolate as well as concentrate trace components with excellent recovery.

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Deep Within the Proteome Milieu, You Can Really Find the Protein That's Right For You

Displacement has been around a long time although a lot of folks still aren’t quite sure what it is and how it’s different than elution chromatography-or what some might refer to as “normal” chromatography.  

So if you are used to elution chromatography, why would you want to change?

Elution chromatography, you know, binding your protein, wash the column, and knock it off in salt, and hope that you get what you want. Sometimes it’s pretty much a trial and error process and prior to the days of recombinant DNA and expression systems for cloning proteins, it could often be a heroic challenge to find that little meaningful thing in your sample. Even with the advent of expression systems for generating recombinant products, often times purity seems to be a relative thing. How pure can you get and how pure is enough? Sometimes when you try to purify something, you lose more than you would like in the process or you end up with something that looks like the protein that you want but in reality is just a little different.

This is where displacement and elution part ways so to speak.  In elution, you typically can only load so much protein and then start your elution. If you overload, the resulting chromatogram is pretty nasty looking. That’s why most manufacturers give you a package insert telling you how much you can load. So while you get sharp peaks, you don’t necessarily get great recovery and if it’s a minor component, you definitely nearly always pay a “protein recovery tax” with each chromatography step until you reach the point of oblivion and you’ve lost all of your protein trying to recover it.

Remember “Isotherms” or maybe you forgot or maybe you just wanted to forget. Well, to make it simple, they represent the binding curves that are specific to each protein when you are trying to purify that protein on a chromatography resin. Well, in elution chromatography, you are in the linear portion of the isotherm curve unless you way overload so your binding is limited as is your recovery when you elute. Now displacement is not operated in the linear range. You load your column up to the max on purpose. You want to be out on the isotherm curve, not in the linear range.

Then the big difference, instead of eluting with salt, which interacts with the resin and protein in an on/off fashion, you add a displacer and the displacer binds to your column. It sounds like affinity chromatography, but not the same. Your components are sequentially “bumped” off in very tight bands-think of them as pure slices. The more of a particular protein, the bigger the slice. The slice is pure protein.

Now the minor proteins. They are separated as well but they are concentrated into little zones adjacent to your major proteins. The recovery of the minor species is excellent as well. The chromatogram is like your teeth before a cleaning. The teeth are the major individual proteins and the minor proteins are the plaque in individual spots between the teeth.

Looking for a biomarker or a needle in the haystack ? Consider displacement. 

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