May 15, 2017 (Vol. 37, No. 10)

Meghaan M. Ferreira Ph.D. Contributor GEN and Clinical OMICs

Proteins Have a Body Language That Depends on Post-Translational Modifications

The cell has a language all its own, one that ultimately rests on a small alphabet of nucleotides, the iconic A, T, C, and G. Somehow, this small alphabet, in accordance with the genetic code, gives rise to a slightly larger alphabet of amino acids, the molecules that combine to form all of the proteins that make up the human proteome.

Moreover, this molecular language, like any language, has additional layers of complexity. Whereas statements in an ordinary language are often articulated or constructed in various forms, depending on the local dialects, the molecular statements that are proteins often acquire subtle shades of meaning—post-translational modifications, for example—that may enhance cell signaling, membrane transport, and other processes.

The messages may be revised in many ways. In fact, there are over 200 different known modification types. They influence protein folding, localization, activation, and other processes. Such modifications are of increasing interest to developers of biotherapeutic proteins. Unlike small molecule drugs, biotherapeutic proteins may vary in terms of safety and efficacy, depending on the messages their post-translational modifications convey. Understanding these messages is a challenge that pharmaceutical companies needn’t confront with small molecules.

Many different types of post-translational modifications exist. After these modifications are identified by mass spectrometry, they can be further scrutinized by antibodies, such as those developed by Rockland Immunochemicals, that can specifically recognize the modified residue and the surrounding amino acids, but not the non-modified sequence or different sequences with the same modification.

Multiple Modification Types

 “If you think about manufacturing a chemical small molecule drug, you realize it’s very simple,” says Laura Moriarty, Ph.D., drug discovery and development marketing manager, Bio-Rad Laboratories. “The drug is manufactured using a chemical synthesis. When creating a biotherapeutic drug, you’re looking at multiple different parameters that can affect the final product.”

Cell lines, growth conditions, purification methods, and drug formulations all have a hand in decorating the final protein with glycosylations, phosphorylations, ubiquitinations, and various other modification types that affect important drug attributes including efficacy, immunogenicity, pharmacokinetics, and degradation.

“Ubiquitination, for example, is a sort of a flag,” explains Poulomi Acharya, Ph.D., senior global product manager at Bio-Rad.  “It says, ‘Hey! Come and degrade me!’” Essentially, ubiquitination changes the half-life of the protein.

Dr. Acharya emphasizes the importance of identifying potential modifications and their effect on each step of drug development, from production and purification to storage and safety.

In the past, protein analysts had to perform complex, multistep procedures to characterize post-translational modifications. “Many people in laboratories would say that these assays take lots of time,” notes Dr. Moriarty. “They’re very hands-on, and they haven’t evolved much over the years.”

The assays may not seem to have evolved, but the interests of protein analysts keep expanding. For example, protein analysts are aware that the types of modifications that need to be targeted have increased, in response to a greater understanding of post-translational modifications and their role in cell signaling. Targeting is about much more than just phosphorylation.

In addition, Biopharma has introduced larger, more complex biotherapeutic molecules that have synthetic modifications, such as PEGylations or, in the case of antibody-drug conjugates, toxic small molecules, in addition to naturally occurring modifications that often increase in frequency with protein size. Meanwhile, more sophisticated mass spectrometry instrumentation and analytical software has enabled researchers to move beyond merely identifying a modification. Researchers may now map a modification’s location on a protein and even quantify the abundance of modified proteins.

A scientist at Bio-Rad Laboratories engaged in antibody validation via high-throughput Western blotting.

Isotopic Labeling

One quantification strategy uses a stable, isotopically labeled peptide that behaves identically to its unlabeled twin, the target peptide, in chromatography and mass spectrometry experiments as an internal standard. Mass differences make it possible to distinguish the otherwise identical pair. To facilitate the use of this method in monoclonal antibody characterization, MilliporeSigma recently introduced SILu™Mab, a product line that provides stable-isotope labeled therapeutic antibodies for mass spectrometry. Researchers can use the relative abundance of the isotopically labeled standard, which has a known initial concentration, to quantify the abundance of the target peptide.

According to Jason Apter, Ph.D., head of research solutions, strategic marketing and innovation, MilliporeSigma, “LC-MS [liquid chromatography used in conjunction with mass spectrometry] has evolved into the predominant method for understanding protein modification, although traditional immunoassay-based methods still exist.”

Mass spectrometry has become the workhorse for protein analytics, but it’s not without its limitations. “The detection of low abundance post-translational modifications is extremely challenging,” explains Dr. Apter, “but is also essential to ensure safety and reproducibility.”

Improved instrumentation has helped overcome detection limitations through increased resolution and mass accuracy, but more abundant peptides can still conceal low concentrations of modified peptides that might contaminate the sample. Many methods exist to enrich samples for the target of interest before mass spectrometry analysis, but the post-translational modification of interest ultimately dictates the techniques available.

Sigma-Aldrich, now a part of MilliporeSigma, provides SILuMAb and SigmaMAb antibody standards for mass spec analysis. This image shows a stable isotope-labeled immunoglobulin G monoclonal antibody. This antibody, which is expressed in a CHO cell line, uses universal heavy-labeled tryptic peptides as internal standards for the quantitation of monoclonal antibodies and Fc-fusion therapeutics.

Additional Enrichment Strategies

Antibody technology can offer researchers more options for enrichment strategies beyond conventional affinity columns and techniques that use radioactive isotopes. For example, BioPlex Pro™ magnetic cell signaling assays, a platform developed by Bio-Rad, uses magnetic beads tethered to phosphorylation state-specific antibodies to isolate phosphorylated proteins of interest. Following immunoprecipitation, researchers can further separate proteins using a Western blot and analyze each resulting band by mass spectrometry to identify the exact modification site on the protein.

The availability of both off-the-shelf and custom antibodies from companies such as Bio-Rad and Rockland Immunochemicals has increased the number of post-translational modifications that researchers can target using immunoassays. Custom antibody collections, such as Bio-Rad’s HuCAL® (human combinatorial antibody library), are the product of recombinant protein technology and in vitro screening methods, such as phage display.

In addition to antibodies that can detect phosphorylation, Rockland Immunochemicals’ catalog includes antibodies that can detect glycosylation, hydroxylation, methylation, acetylation, and ubiquitination. Antibody technology, given its relative cost-effectiveness, ease of use, and platform-independence, has come to be tightly embraced by the research community, remarks Carl Ascoli, Ph.D., Rockland’s chief science officer. Dr. Ascoli foresees a future in protein characterization that has “a good blend of both antibody technology and mass spec technology.”

Multiple Attribute Monitoring

While the advent of PCR has driven progress at the nucleic acid level, “mass spectrometry in the biopharmaceutical or biotech realm has really been the driver from an analytical standpoint,” according to Glenn Petrie, Ph.D., senior scientific advisor at EAG Laboratories.

EAG Laboratories is a contract research organization (CRO) that works with Biopharma and Biotech clients to provide comprehensive analytical characterization and GMP testing of biotherapeutic proteins (including monoclonal antibodies and antibody-drug conjugates). “[To characterize] PTMs in the old days, you would have had to use multiple methods,” explains Rowel Tobias, Ph.D., principal scientist and group lead, EAG Laboratories. “With high-resolution mass spectrometry, you can combine all of this into one MAM (multiple attribute monitoring) method.”

EAG Laboratories uses quadrupole time-of-flight (q-TOF) and QTRAP mass spectrometers to perform their characterizations. These high-end instruments have high resolution and mass accuracy and are able to detect post-translational modifications present at low levels in the sample. These advanced capabilities come with a high price tag, though.

“These instruments start at a half a million [dollars] and then go up,” admits Dr. Petrie. “It’s a big leap in both cost and technology, as well as the expertise that’s required to run these sorts of analyses.”

However, for Biopharma companies looking to produce biosimilars, the trade-off between providing comprehensive comparisons between a biosimilar and an innovator molecule and having to perform additional clinical trials makes the additional cost for analyses a worthwhile exchange. It’s one reason the emerging biosimilar market has driven the field of protein analytics tremendously.

Nonetheless, as technology becomes more advanced and robust, the FDA continues to increase its expectations. “They want you to use the current and most powerful tools to provide more detailed information than before,” comments Dr. Petrie.

Quality considerations set forth by the FDA also highlight the necessity of studying biotherapeutics under the same environmental conditions the protein will face during manufacturing and storage, and even during its time in the body. Chemical modifications that can result in response to environmental factors, such as oxidation and deamidation, can elicit unwanted immune responses to the drug.

EAG Laboratories uses high-res MS to help its clients characterize biotherapeutics, which can be hard to analyze if they carry synthetic add-ons. (This antibody-drug conjugate could harbor several distinctive features.)

Hyper-Reaction Monitoring

Biognosys, a Switzerland-based company that specializes in developing and providing proteomics technology, uses a novel workflow based on high-resolution mass spectrometry called hyper-reaction monitoring (HRM). In contrast to classical proteomics methods, where each peptide is analyzed sequentially, HRM fragments all peptides together, which “provides deep and comprehensive data,” according to Biognosys’ co-founder and CEO, Oliver Rinner, Ph.D. “HRM is especially suited for very complex samples, but also for samples with very large differences in protein quantity, like biotherapeutics, where certain modifications occur only in very low stoichiometry.”

Whereas Biognosys’ main focus is on analyzing complex proteomes, they also work with isolated proteins—characterizing the presence of modifications and identifying “breaking points” that make the protein susceptible to oxidation and other degradation pathways.

One of the things Biognosys does very well, asserts Dr. Rinner, is facilitate comparisons across conditions. For example, the company’s technology can help researchers “test certain differential conditions, like storage conditions.” Biognosys has also developed a kit for determining indexed retention time (iRT), an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction, and signal processing software (Spectronaut™) that can deconvolute the highly dimensional data HRM produces.

Hyper-reaction monitoring (HRM) is a technology based on high-resolution mass spectrometry that can be used to quantify post-translational modifications. This image, provided by Biognosys, highlights portions of the HRM workflow (building a spectral library, acquiring data in DIA mode, and analyzing data) that can be supported by proteomics technology, namely, Biognosys’ sample preparation kit, indexed retention time (iRT) kit, and Spectronaut software.

Data Analysis

It takes the “right type of software tools” to handle the terabytes of data that mass spectrometry analyses generate, asserts Eric Carlson, Ph.D., president and CEO at Protein Metrics. “Without the right type of software tools, the analysis would really be left to a small handful of individual experts. Having the right software tools really allows this type of analysis to be brought mainstream.”

Protein Metrics offers a suite of analytical modules that work with their search engine, Byonic™, to enable protein characterization: Byologic®, Byomap™, and Intact Mass™. Byologic enables detailed inspection of mass spectra. Using this module, analysts can characterize proteins and their post-translational modifications and other variants in greater detail, compare multiple samples, and perform label-free quantification.

Byomap allows analysts to look at data obtained from both mass spectrometry and chromatography instruments, such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). The software module can generate a reference standard to identify and annotate different chromatographic peaks using mass spectrometry data to simplify downstream processes, like lot release.

Finally, Protein Metric’s Intact Mass software analyzes mass spectra of undigested molecules, often referred to as the “top-down” approach to protein analytics. Top-down analysis is a much faster technique that requires less sample prep than the alternative “bottom-up” method. But if there is a post-translational modification on the molecule, this method does not allow localization.

Protein Metric’s software is vendor-independent, which creates continuity and consistency for pharmaceutical companies with multiple instruments from different vendors. “All the reports can look the same, and all the methods can look the same,” stated Dr. Carlson. “That’s how we can be very consistent. Analysts can spend their time focusing on the samples—as opposed to developing new methods that work only on their instruments.”

Mass spectrometers and separation technologies have incorporated advances that improve resolution and thereby enable researchers to see small, subtle differences between samples. Although these advances make datasets more difficult to analyze, Dr. Carlson views them as “an opportunity for a software company to really be able to provide data analysis for these more complicated datasets.”

As scientists continue to intercept and decipher the messages that post-translational modifications send, biopharmaceutical companies will have an opportunity to translate these discoveries into better drug design and development—but it all relies on the analytical methods and tools available to interpret the language of proteins.

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