SACHEM Displacement Chromatography

SACHEM Launches Lines of Reversed-Phase Displacers for Protein Purification

SACHEM now has available RP reversed-phase displacers, an enabling line of displacers for the analytical or preparative purification of peptides, denatured proteins, neutral, cationic or anionic drug intermediates as well as oligonucleotides (DNA or RNA). Below is an overview of available displacers for specific applications. For questions and further information, please contact SACHEM's Biotech team.

"Isolation of Monoclonal Antibody Charge variants by Displacement Chromatography” Paper Published in Current Protocols in Protein Science, Aug 2012 Issue

Current Protocols in Protein Sciences published a new article on the Isolation of Monoclonal Antibody Charge Variants by Displacement Chromatography.

Authors Dr. C. Patrick McAtee and Jacob Hornbuckle discuss the important parameters in designing and optimizing a separation of monoclonal antibody (mAb) charge variants from process streams by ion-exchange displacement chromatography.

The protocol also reviews sample preparation and selection of matricx, column, and appropriate buffer. Monoclonal antibody (mAb)-directed therapeutics represent the vast majority of new drug development programs in the pharmaceutical and biotechnology industry today.

The European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have recently published new guidelines calling for enhanced scrutiny of mAbs, particularly with regard to analytical data, which could affect product pharmacokinetics (PK) and immunogenicity. This guidance has become most relevant in the realm of biosimilars or “biobetters.”

"As manufacturing guidelines become increasingly more rigorous with a focus on detailed analytical parameters, this technology offers a new and insightful methodology for evaluation of factors contributing to the safety and efficacy of biotherapeutics. This represents a novel approach for analysis and production of biological therapeutics as it offers the ability to enhance purity to levels previously unattainable, which can affect PK profile and performance. A biosimilar so purified becomes a “biobetter”. ” commented Dr. Patrick C. McAtee, Sr. Manager of Biotechnology Applications at SACHEM.

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

Monoclonal Antibody Characterization

Charge-related heterogeneity is a common issue in nearly every monoclonal antibody process. Purification  and elimination of these isoforms can be somewhat daunting. Using displacement chromatography reagents, you can isolate , purify, and even eliminate these charge variants.

Displacement Chromatography - A QbD Platform for the Analysis of Biological Therapeutics

Biological therapies are rapidly gaining acceptance in the treatment of a wide spectrum of diseases. While many complex diseases such as rheumatologic disorders are routinely addressed by biologics, applications to non-rheumatic diseases such as psoriasis, inflammatory bowel disease, diabetes, and multiple sclerosis are currently in development. (Bell et. al. 2011). However, on some occasions, treatment regimens can lead to paradoxical effects such as an actual worsening of the disease.

The mechanism behind this unexpected outcome has been documented in at least two studies where biological therapy was administered. In the first example, antibody associated pure red cell aplasia was discovered in patients who had undergone treatment with recombinant human erythropoietin (EPO).   (Bennett et. al. 2004) It was determined that a convergence of factors related to production, handling, and route of administration may have accounted for the EPO associated pure red cell aplasia. Processes and formulations that facilitate oxidation or aggregation of the protein can enhance immunogenicity. In another example, TGN1412 was an anti-CD28 Monoclonal antibody (MAb) initially developed for the treatment of B cell chronic lymphocytic leukemia (B-CLL) and rheumatoid arthritis. In its first human clinical trials, it caused catastrophic systemic organ failure in the subjects, despite being administered at a supposed sub-clinical dose of 0.1 mg per kg; some 500 times lower than the dose found safe in animals (Coghlan 2006). Six patients presented evidence of “cytokine storm”, a potentially fatal immune reaction consisting of a positive feedback loop between cytokines and immune cells (http://en.wikipedia.org/wiki/Cytokine_storm) , with highly elevated levels of various cytokines (Suntharalingam et. al. 2006). While physicians at the time declared that it was impossible to anticipate such severe reactions, particularly in the light of convincing  animal safety data, one has to wonder  whether a particular variant of TGN1412 may have been the culprit and in fact, this has led to a proposal for guidelines on immunogenicity of biotherapeutics in the EU.

Although MAbs present a different set of challenges from other product classes, the level of risk associated with first administration to humans can be effectively mitigated through appropriate pre-clinical strategies addressing product safety.  The analysis of contributing risk factors for safety must include a thorough analytical profile of the product. Product aggregates and process related impurities could alter immunogenic potential. Further, the presence of product variants that have different biological activities could significantly influence the overall benefit-to-risk for humans (Chamberlain 2011) .

Most approved biopharmaceuticals have shown some immunogenicity although the percentage of patients developing antibodies upon treatment with protein-based drugs varies widely. Factors influencing the immunogenicity of proteins are manifold and include amino acid variations, modification of amino acid side chains, variation in glycosylation, as well as the presence of unseen contaminants.  It is therefore imperative that manufacturers identify and hopefully eliminate these issues early in development. Apart from the need for a consistent manufacturing process, immunogenicity must be monitored-either directly or by a reliable surrogate method. 

The use of displacement chromatography provides a platform for analysis of biological therapeutics and has been used to identify charge related variants in an IgG1 MAb preparation ( Zhang et. al. 2011). In this paper , Genentech scientists demonstrated the value of including displacement chromatography in MAb characterization and highlighted  the potential to greatly enhance the information available regarding the development of complex biological therapeutics. As a preparative technique, cation exchange displacement chromatography allowed for significant enrichment of charge variants with high purity and recovery. Using displacement, one can generate a ‘fingerprint” of each processed batch based upon presence of variants encountered during manufacturing. This fingerprint can provide useful information to help correlate product efficacy with manufacturing protocol and could possibly help compensate for lack of predictability of human immunogenicity based on animal studies alone.

REFERENCES:

Bell, et. al Nature Reviews in Rheumatology (2011) 7: 507-516

Bennett, et. al. New England Journal of Medicine (2004) 351: 1403-1408

Chamberlain New Biotechnology (2011) 10.1016: 1-8

Coghlan, New Scientist (2006) 12: 14-16

Suntharalingam, et. al. New England Journal of Medicine (2006) 355: 1018-1028

Zhang et. al. Journ. Chromatog A (2011) 1218: 5079-5086

 

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 

 

Knowledge is Power...Displacement Chromatography Provides Knowledge

With displacement chromatography, you have the ability to better understand your processes. This is going to allow you to make better decisions,  improve your products, create better processe paths...and help more patients.

Don't be afraid of the uncertain...understand what you have...

Read the recently published article to learn how Genetech was able to identify trace components they were not able to see before. Research scientists Taylor Zhang, Justin Bourret and Tony Cano demonstrated the isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography.

Journal of Chromatography A
Volume 1218, Issue 31, 5 August 2011, Pages 5079-5086

 

mAb Characterization | How Does Displacement Chromatography Compare to Elution?

Displacement chromatography is an ideal chromatographic purification technique to identify and isolate mAb charge variants. Why? The technique has the capability to isolate minor components that you can't see with most other methods. This is particularly beneficial for the analysis, production and purification of monoclonal anitbodies in drug therapy development. With displacement chromatography, it is possible to  identify and isolate charge variants. Through the idenfication of minor components, you can then identify which variants affect the characteristics the drug. This is particularly helpful for Quality by Design and quality assurance. (FDA guidance)

How does displacement compare to and differ from elution chromatography?

Displacement chromatography:

• Purifies principal components but also simultaneously concentrates minor components and impurities
• Yields selective enrichment of trace components in complex matrices
• Allows for detection of low-level impurities or close-runnig impurities that are otherwise unobservable by other purification methods (ppm level)
• Uses standard laboratory equipment (i.e. analytical HPLC)

How does displacement differ from elution? The images below illustrated similarities and differences. If the tow images below don't display, please click here for the pdf slides.

 

“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.

Reduce Uncertainty and Unwanted "Surprises" in Process Scale-Up

Source: "Ultrasensitive Impurity Profiles by Displacement Chromatography (American Biotechnology Laboratory Oct 2007)

Displacement chromatography with displacers (i.e. Isolis™)  is effective in increasing the detection sensitivity in impurity profiles. Why would this matter?

In method transfers and scale-up, the impurity profile of the new and reference process are often compared. Often process design and control strives for consistency; thus any change is undesirable. This is especially true in purification schemes involving multiple units or steps.

A typical impurity profile of a mixture of ß-lactoglobulin A and B by ion exchange chromatography is presented in the following figure 1.
It shows two main peaks and four, perhaps five, minor peaks. This is typical of impurity profiles reported for many proteins, including therapeutics. The low level of impurities builds confidence that the purification scheme has worked well.

Displacement chromatography can be used to purify proteins, as shown in the histogram below. Conventional HPLC runs are complete in about one hour. A DC study of impurity profile is a two-step process. The first step is running the DC, including a collection of appropriate fractions. The second step is analysis of the fractions, which can be time consuming.

The center cuts of the DC give 85-86% recovery of a 99.9% pure products. This can be useful in laboratory and process scale preparation. However, DC also concentrates the impurities in the regions between the major peaks (see A-F). Analysis of the fractions from DC shows that these contain 23 impurities. Twelve of the peaks have been identified as related lactoglobulins, and 11 are unknown impurities.

It is important to note that the vast majority of the peaks in the following table were not seen in the impurity analysis shown in Figure 1.

Displacement chromatography increases the peak count in impurity profiles by threefold, and the measured level of impurities by about 33%.

Read the complete technical article in American Biotechnology Laboratory:

Ultrasensitive Impurity Profiles by Displacement Chromatography: A Comparative Study