October 15, 2017 (Vol. 37, No. 18)

Emphasizes a Combination of Antibody-Dependent and -Independent Testing Methods

The structure of antibodies is well known, but understanding their true function is where things get more nebulous. [knorre/Getty Images]

Authentication is becoming a hot-button topic among researchers. For example, researchers who use cells that are meant to possess distinct characteristics have been making an issue of cell-line authentication.1 With increasing frequency, researchers who use antibodies are raising authentication concerns of their own. These researchers are insistent that antibodies be validated for function and purpose.

Antibodies are critical research reagents, says Nicolas Schrantz, Ph.D., senior manager for antibody development at Thermo Fisher Scientific. They can lead to research success, but only if they perform as desired—which is to say, as advertised. “Developing an antibody that recognizes the correct target using a specific application is an art that requires scientific knowledge and technical expertise,” notes Dr. Schrantz. “Unfortunately, many antibodies on the market that claim to have been validated for a specific application simply do not recognize the target they are supposed to, or worse, recognize the wrong target. The existence of unreliable antibodies wastes researchers’ time and money.” 

Two-Step Validation

Thermo Fisher Scientific has been working with individual researchers and the International Working Group for Antibody Validation (IWGAV) to define and implement standards for antibody testing. The company has already adopted validation standards for its Invitrogen antibody portfolio that adhere to IWGAV recommendations.

Thermo Fisher Scientific uses a two-step validation approach.  The first step is target specificity verification. The second step is functional application verification.

Target specificity verification assures that antibodies bind to the correct target. Validating an antibody’s specificity also ensures the absence of nonspecific binding.

To accomplish target specificity validation, Thermo Fisher uses at least one standard method from a collection of standard methods. These methods span several categories: immunoprecipitation/mass spectrometry; genetic modification (knockout and knockdown testing); independent antibody verification (a testing approach that uses two differentially raised antibodies that recognize the same protein target); and biological verification (cell treatment, relative expression, neutralization, peptide array, and orthogonal approaches).

Thermo Fisher employs several knockdown and knockout methods to test antibody performance against genetically modified samples. They include mouse knockout models, dominant negative mutants, morpholinos, short interfering RNA, and most recently, gene editing. CRISPR/Cas9 gene editing allows creation of knockout cell models for use as controls for validating antibody specificity.

In independent antibody verification, antibodies should target non-overlapping epitopes on an antigen. “Obtaining comparable [affinity] results increases confidence that these antibodies are specific and suitable for the detection of their intended target,” Dr. Schrantz tells GEN.

With the orthogonal approach, the idea is to correlate two methods, an antibody-dependent method and an antibody-independent method. For example, Western blot could be correlated with quantitative RT-PCR, or flow cytometry could be correlated with Thermo Fisher’s own PrimeFlowRNA assay kit.

The second level of validation involves determining how well antibodies work in applications such as Western blotting, immunofluorescent imaging, flow cytometery, chromatin immunoprecipitation, and immunohistochemistry. “We test our antibodies using at least one of these methods,” states Dr. Schrantz.“And yes, we even use antibodies or kits from other vendors for this purpose.”

Middle-Up Approach

Monoclonal antibodies (mAbs) are inherently heterogeneous because they are produced in living cells and can undergo unanticipated modifications during the biomanufacturing process. Living cells are capable of variability at every stage of protein expression, from the generation of amino acid chains during the formation of protein backbones, to the introduction of post-translational modifications (PTMs), some of which occur enzymatically (glycosylation), and some of which occur non-enzymatically (oxidation and deamidation). Additional modifications may occur during purification and storage.

GEN readers are familiar with chromatographic and compound analytical methods, as well as the importance of using multiple, sometimes orthogonal methods, for example, size-exclusion or ion-exchange chromatography and peptide mapping. Peptide mapping followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS)—the method of choice for understanding site-specific PTMs—involves serious sample preparation and lengthy chromatography runs. Researchers refer to proteolysis-based mapping as bottom-up proteomics, and those based on intact proteins as top-down methods.

Peptide mapping is a multistep process generally involving antibody denaturing, reduction, and alkylation; digestion with a protease (usually trypsin); high-performance liquid chromatography (HPLC) on a octadecyl carbon chain (C18)-bonded silica column; and finally, mass spectrometry “online” with HPLC to identify the separated peptides. Together, the steps in peptide mapping take about one full day, including overnight digestion.

Peptide map data analysis is complex by virtue of the sheer number of peptides generated, all of which require identification and quantitation. Analysis gets even trickier when full digestion does not occur, or when products of nonspecific digestion are present.

Investigators involved in mAb characterization are therefore interested in alternatives to full peptide mapping. Many of these methods, including middle-up or middle-down, employ proteolytic enzymes that are somewhat more selective than trypsin.

Among these is the immunoglobulin-degrading enzyme from Streptococcus pyrogenes (IdeS), which was first reported by Swedish researchers in 2002,2 and is now provided in commercialized in kit form by several vendors. IdeS reagents and kits are sold by Genovis, MilliporeSigma, Creative Enzymes, and Promega. “IdeS is effective due to its high cleavage specificity and simple operation,” explains Chris Hosfield, Ph.D., a senior research scientist at Promega. A related enzyme, IdeZ (from S. equi subspecies zooepidemicus), has identical specificity but cleaves mouse IgG2a more efficiently than IdeS.

IdeS cleaves the IgG heavy chain below the hinge region. Post-digestion addition of a reducing agent yields a sample containing three fragments of about 25 kDa in size. This is the starting point for subsequent analysis—hence the “middle-up” designation. The approach identifies domain-specific oxidation, charge profiling, and N-glycan profiles. In a study published in MAbs,3 domain separation was achieved with a 30-minute HPLC gradient, and oxidations were quantified through ultraviolet detection.

IdeS is faster than peptide mapping, taking up to about one hour instead overnight. The separation stage is faster because only three fragments are involved. “HPLC gradients are in the range of 30 minutes instead of 3 hours,” asserts Dr. Hosfield. Data analysis is simpler for the same reasons. “For these reasons, many companies use IdeS-based analysis as a platform method, for example, in quality control lot-release settings.”

The downside is that information on modifications is not site-specific. For example, one could detect an oxidation occurring in the Fc region, but not determine definitively which methionine within that region was affected.

A more recent study by a group at Genentech described an improved IdeS in which separation of the IgG domains and variants took just 10 minutes.4 Measured oxidation levels were comparable to those achieved by more complex and time-consuming peptide mapping.

Putting Validation to Use

The level at which manufacturers validate antibodies depends on the antibodies’ intended use. An example of how validation level may complement antibody use is provided by Abbiotec, which manufactures both polyclonal antibodies (pAbs) and mAbs for research purposes. The company characterizes its products for target reactivity, but according to CEO Hervé Le Calvez, Ph.D., it doesn’t assay products for physicochemical properties, outside of purity determinations by ELISA or SDS-PAGE, which specifically test for contamination by other antibodies.

“Our approach is similar between pAbs and mAbs, especially when we use peptides as antigens,” Dr. Le Calvez says. “We mainly produce GLP-grade antibodies, so we make sure screening, titering, and isotyping, if applicable, are done correctly before moving on with purification and testing.” Purification methods for pAbs vary from standard Protein A or Protein G to antigen-affinity chromatography and fractionated precipitation.

“Because of the difficulties in characterizing pAbs from each bleed or animal,” explains Dr. Le Calvez, “we offer the option to clone the genes coding for the antibodies and produce recombinant antibodies when the end goal is the diagnostics or therapeutics market.”

One issue entering the validation/characterization equation is the antibody target. “It’s one thing to validate an antibody against a well-known target such as TNFα [tumor necrosis factor alpha] or NF-?B [nuclear factor kappa B], but quite another if the target is the latest new protein identified by sequencing,” insists Dr. Le Calvez. “Our products lean towards the latter.”

There is much to learn, he adds, about the validation of antibodies that target new molecules that have not themselves been fully characterized. Abbiotec releases such products as a service to research groups that do not fully disclose their intentions. “In this respect,” reveals Dr. Le Calvez, “we provide numerous antibodies for niches that are still undeveloped.”

Lest We Forget…

In the characterization of intact, therapeutic-grade mAbs, conventional analysis tools are being deployed more systematically. As mass spectrometry (MS) becomes more user-friendly and generally accessible, mAb developers routinely adopt this method to obtain a product’s accurate molecular weight and heterogeneity (for example, with respect to PTMs). For development-stage antibody-drug conjugates, MS provides further insights into conjugation number, mAb sequence variations, and degradation products.

“Confidence in the information obtained from analyzing intact mAbs depends on measurement reliability,” explains David Wong, Ph.D., a senior applications scientist at Agilent Technologies. “Accurate mass determination, separation of protein isoforms, and detection of major and minor heterogeneities provides reliable answers about the protein and growth conditions directly from intact protein analysis.”

Due to its high resolution in high mass ranges, quadrupole time-of-flight (Q-TOF) LC-MS is the mode of choice for analyzing intact proteins. Top vendors have “systematized” these instruments toward specific purposes. For example, the Agilent 6545XT AdvanceBio LC/Q-TOF system includes hardware and software features for characterization of biomolecules up to 30,000 m/z.

“Q-TOFs have the flexibility to analyze not just intact proteins, but also to perform peptide sequence mapping and PTM identification and localization at the peptide level,” Dr. Wong adds.

In a recent application note,5 Agilent described a typical workflow involving the Agilent 1290 Infinity II UHPLC system (at the front end of separation), the AdvanceBio LC/Q-TOF, and the company’s MassHunter BioConfirm software (for automatic data processing). The analyte was a NIST [National Institute of Standards and Technology] mAb standard.

LC-MS analysis showed mass resolution of all species falling between 2,000 and 5,000 m/z. Moreover, zoom-in spectra of each charge state showed the six major glycoforms of the NIST mAb. In addition to these major features, the analysis identified minor glycosylation heterogenicities, such as loss of N-acetylglucosamine (Figure).

Agilent analysis software includes a maximum entropy deconvolution algorithm, which preserves fine details of the intact protein. Typically, a mAb can have 30–70 positive charges under LC-MS conditions. Consequently, a mAb with a nominal mass close to 150,000 amu is generally detected in the range of 2,000–5,000 m/z.

According to Dr. Wong, the maximum entropy algorithm has been widely used for deconvolving the multiple charge state envelope that occurs when a protein is analyzed by LC-MS: “The method transforms the raw m/z spectrum of one or more intact proteins into the actual molecular weight of the protein, making it easier to directly compare [it] to theoretical sequence information or potential mass shifts caused by PTMs.”

Figure. Major glycoforms and minor isoforms alike are clearly seen with the Agilent 6545XT AdvanceBio LC/Q-TOF system. The data was deconvoluted using the maximum entropy algorithm in the Agilent MassHunter BioConfirm software. This algorithm carefully preserves low-level peaks so that the heterogeneity of a molecules may be fully characterized.

1. A. DePalma, “Cell-Line Denial Is Too Costly, Authenticate!,” GEN 36(20), 24–27 (2016).
2. U. von Pawel-Rammingen, B.P. Johansson, and L. Björck. “IdeS, a Novel Streptococcal Cysteine Proteinase with Unique Specificity for Immunoglobulin G,” EMBO J. 21, 1607–1615 (2002).
3. Y. An, Y. Zhang, H.M. Mueller, M. Shameem, and X. Chen, “A New Tool for Monoclonal Antibody Analysis: Application of IdeS Proteolysis in IgG Domain-Specific Characterization,” MAbs 6(4), 879–893 (2014).
4. B. Zhang, J. Jeong, B. Burgess, M. Jazayri, Y. Tang, Y. Taylor Zhang, “Development of a Rapid RP-UHPLC-MS Method for Analysis of Modifications in Therapeutic Monoclonal Antibodies,” J. Chromatog. B Analyt. Technol. Biomed. Life Sci. 1032, 172–181 (2016).
5. D.L. Wong, “Precise Characterization of Intact Monoclonal Antibodies by the Agilent 6545XT AdvanceBio LC/Q-TOF,” Agilent Technologies, publication number 5991-7813EN (application note).

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