Analysis of a biosimilar mAb using Mass Spectrometry

Several pharmaceutical companies have biosimilar versions of therapeutic mAbs in development. Biosimilars can promise significant cost savings for patients, but the unavoidable differences
between the original and thencopycat biologic raise questions regarding product interchangeability. Both innovator mAbs and biosimilars are heterogeneous populations of variants characterized by differences in glycosylation,oxidation, deamidation, glycation, and aggregation state. Their heterogeneity could potentially affect target protein binding through the F´ab domain, receptor binding through the Fc domain, and protein aggregation.

As more biosimilar mAbs gain regulatory approval, having clear framework for a rapid characterization of innovator and biosimilar products to identify clinically relevant differences is important. A recent reference (1) applied a comprehensive mass spectrometry (MS)-based strategy using bottom-up, middle-down, and intact strategies. These data were then integrated with ion mobility mass spectrometry (IM-MS) and collision-induced unfolding (CIU) analyses, as well as data from select biophysical techniques and receptor binding assays to comprehensively evaluate biosimilarity between Remicade and Remsima.

The authors observed that the levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. they found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact.  Importantly, they identified more than 25 glycoforms for each product and observed glycoform population differences.

Overall the use of mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. They demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

1. Pisupati, K. et. al. (2017) A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Anal. Chem 89, 38–46.

Don’t Let These Three Common Issues Hurt Your Luminescent Assay Results

4621CAThere is a lot riding on your luminescent assay results. Each plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate?  Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for.  Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work.  Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments. Continue reading

Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed for characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.

We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process, reducing the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.

How Does it Work?

With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.

Continue reading

Gut Microbes and Hypertension: Demonstrating a Causal Link

Most of us are aware that the human body is covered by and full of microorganisms. And we understand that most of these microorganisms are helpful, both in terms of competition with and protection against invading microorganisms, and in the gut, as agents of digestion.

Bacillus subtilis, an example of Firmicutes, and not a good gut microbe. By Y tambe (original uploader) - Own work, CC BY-SA 3.0,

Bacillus subtilis, an example of Firmicutes, and not a good gut microbe. By Y tambe (original uploader) – Own work, CC BY-SA 3.0,

In the past decade, however, research has brought compelling details implicating gut microbes in obesity, cancer, insulin resistance and such central nervous system disorders as depression, austism spectrum disorder and multiple sclerosis (Adnan, S. et al.). Yet the mechanisms and details of these associations have not been fully demonstrated.

Gut bacteria have been proven to be connected to thickening of heart vasculature, known as atherosclerosis. Researchers have demonstrated that bacteria metabolize choline and L-carnitine from food to trimethylamine, which crosses the gut barrier into circulation and reaches the liver. In the liver, trimethylamine is metabolized to the atherogenic molecule triethylamine-N-oxide (Gregory, J.C. et al., Brown and Hazen). These studies are among the few that provide a direct connection between gut microbes and a pathological condition. Continue reading

The Wide World of Bioprocessing: Science for the Greater Good

My former research career was spent in academic laboratories, and I don’t have first-hand experience in the world of bioprocessing. However in my current job as a science writer/copy editor, I create product information and literature about products that are useful to bioprocessing engineers and technicians, and thus wanted to learn more about this diverse area, where discovery and processing of biomaterials results in better therapeutic drugs, better biofuels and even healthier foods.

Bioprocessing is a combination of biological science and chemistry, and a burgeoning science field. Burgeoning is an understatement. Exploding is a much more apt description.

This 2011 Science magazine careers article defines bioprocessing thusly:

“Bioprocessing is an expanding field encompassing any process that uses living cells or their components (e.g., bacteria, enzymes, or chloroplasts) to obtain desired products, such as biofuels and therapeutics.”

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Improved Method for the Rapid Analysis of Monoclonal Antibodies Using IdeS

ides_abTherapeutic monoclonal antibodies (MAbs) are inherently heterogeneous due to a wide range of both enzymatic and chemical modifications, such as oxidation, deamidation and glycosylation which may occur during expression, purification or storage. For identification and functional evaluation of these modifications, stability studies
are typically performed by employing stress conditions such as exposure to chemical oxidizers, elevated pH and temperature.

To characterize MAbs, a variety of analytical techniques are chosen, such as size exclusion chromatography and ion exchange chromatography. However, due to the large size of the intact MAbs, these methods lack structural resolution. Often, the chromatographic peaks resolved by SEC and IEC methods are collected and further analyzed by peptide mapping to obtain more detailed information. Peptide mapping, in which antibodies are cleaved into small peptides through protease digestion followed by LC–MS/MS analysis, is generally the method of choice for detection and quantitation of site-specific modifications. However sample preparation and lengthy chromatographic separation make peptide mapping impractical for the analysis of large numbers of samples. In contrast to peptide mapping analysis, the middle-down approach offers the advantage of high-throughput and specificity for antibody characterization.

Limited proteolysis of IgG molecules by the IdeS enzyme has been introduced for antibody characterization due to its high cleavage specificity and simple digestion procedure. Continue reading

Optimizing Antibody Enrichment for Pharmacokinetic Assays

Schematic showing immuno-enrichment using High Capacity Magne® Streptavidin Beads.

Schematic showing immuno-enrichment using High Capacity Magne® Streptavidin Beads.

During preclinical research and development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and primates. Ligand-binding assays (LBA) or liquid chromatography coupled to tandem mass spectrometry(LC–MS/MS)-based methods represent the two most common technologies used to perform the PK studies for mAb candidates(1,2).

Using either method it is essential to ensure accurate quantitative results that the initial enrichment of the target therapeutic antibody from serum or plasma be optimal. Biotinylated antibodies or antigens (against the therapeutic targets) immobilized onto high capacity streptavidin beads will enrich therapeutic antibody from serum or plasma samples. (Figure13666MC.eps). The affinity of biotin for streptavidin (Kd = 10–15) is one of the strongest and most stable interactions in biology therefore the biotin-streptavidin interaction cannot be reversed under non-denaturing conditions. Hence, it is possible to perform extensive washing to remove nonspecifically bound protein and elute therapeutic antibodies without also eluting the biotinylated component, thus improving the detection limit.

Magnetic based separation techniques have several advantages in comparison with standard separation procedures. This process is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one single test tube. The magnetic separation techniques are also the basis of various automated procedures. Learn more about  the High Capacity Magne™ Streptavidin Beads (Cat # V7820) .


Shooting for the Moon: Better Assays to Hit Our Cancer Research Targets

3239CA02_1AIn his address to the clinicians, researchers, and patients at the American Association for Cancer Research meeting in April, US Vice President Joe Biden, revealed that the goal of the #cancermoonshot initiative is to accomplish 10 years of cancer research in just five years, effectively doubling the pace of cancer research (1).

Treatments developed from cancer research have come a long way with dramatic differences in the experiences and prognoses for patients, just looking back over the last 25 years. How can we double the pace of cancer research? The #cancermoonshot will one, encourage data sharing among researchers, particularly data from clinical trials. Second, it seeks to increase collaboration across industry, academic and government scientists—each community being positioned to make unique contributions to the field. And third, the initiative looks to change the current grants award process that encourages scientists to keep data and results “quiet” until they can be published or protected legally as intellectual property.

Immunotherapy is an especially hot field in cancer research (2) that relies on the immune system to better fight cancer. Continue reading

Optimizing Antibody Internalization Assays: pHAb Dyes

22255190_pHabAmine_3D_image_050615-final-largePromega has recently developed a method that allows antibodies to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When an antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane, the antibody-dye-antigen complex is not fluorescent, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops, and the dye becomes fluorescent.

To demonstrate the broad utility of the pHAb dye for receptor mediated antibody internalization, two therapeutic antibodies, trastuzumab and cetuximab,which bind to HER2 and EGFR respectively, were selected for a case study (1). Both the antibodies, which are known to internalize were labeled with pHAb dyes using amine or thiol chemistry.

Parameters such as the impact of dye–to-antibody ratio on the antigen–antibody binding, change in fluorescence as a function of pH of free dye and labeled dye, and labeled antibody internalization as a function of pHAb conjugated antibody concentration were evaluated.

The results indicate that pHAb dyes are pH sensitive fluorescent dyes that enable the study of receptor-mediated antibody internalization.Internalization assays can be performed in a plate-based homogeneous format and allow endpoint assays as well as real-time monitoring of internalization. They further show that internalization can be monitored even at a very low amount of antibody which is very important during the early monoclonal antibody development phase when the amount of sample is limited and the antibody concentration in the samples is low. a complimentary approach, they  also showed that a secondary antibody labeled with pHAb dye can be used instead of labeling primary antibodies.

Literature cited

Nath, N. et al. (2016) Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye J.  Immunol. Methods epub ahead of print

Characterizing Immune-Modulating Antibodies Using Bioluminescence

Immune checkpoint pathways such as PD-1/PD-L1 and CTLA-4 are promising new immunotherapy targets for the treatment of cancer and autoimmunity. Immune checkpoint reporter-based bioassays provide a simple, consistent, and reliable cell-based assay to measure Ab function throughout the drug development pipeline.

The brief chalk talk below describes the assay principals of the reporter-based bioassay that monitors the functional blockade of PD-1/PD-L1 interactions.