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Most read from mAbs

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The Antibody Society is pleased and proud to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue. All the articles are open access; PDFs can be downloaded by following the links below.

Issue 10.8 (November/December 2018)

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination. In this new report, Resemann et al. discuss a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. They established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms, and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis. Bailey et al. describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. They used pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. The authors demonstrated that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Based on their results, they conclude that the CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. In this new report, Wang et al. discuss a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

Characterization and analysis of scFv-IgG bispecific antibody size variants. Cao et al. report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, the authors developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

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Most read from mAbs issue 10.6

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The Antibody Society is pleased and proud to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these summaries based on the abstracts of the most read papers published in a recent issue. All the articles are open access; PDFs can be downloaded by following the links below.

Issue 10.6 (Aug/Sep 2018)

Antigen recognition by single-domain antibodies: structural latitudes and constraints. In this new review, Henry and MacKenzie comprehensively surveyed the evidence in support of the hypothesis that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. They found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, the authors conclude that sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.

Biosimilars in oncology and inflammatory diseases: current and future considerations for clinicians in Latin America. Scheinberg et al. review the use of biosimilars in Latin America, which is complicated by the presence of “non-comparable biotherapeutics” (also known as “intended copies”) that have not been rigorously compared with the originator product. The authors discuss the current situation and the considerations for clinicians in Latin American countries, focusing on monoclonal antibody biosimilars relevant to oncology, rheumatology, gastroenterology, and dermatology.

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies. In this new report, Jetha et al. optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. This analysis was extended to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and the authors found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.

Heterologous recombinant expression of non-originator NISTmAb. Kashi et al. describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. The authors found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. SDS-PAGE, SEC, NMR spectroscopy, intact MS, and SPR were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.

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We encourage you to join the Society to take advantage of the substantial benefits of membership, including discounts on fees for selected KNect365, CHI, and Hanson Wade meetings, discounted subscriptions to Society-affiliated journals PEDS and mAbs (special subscription rate of US $84 online only access for Antibody Society members)  and access to information in the Members Only section of the website. In particular, we encourage members to take advantage of the discount on registration for Antibody Engineering & Therapeutics, which is the annual meeting of The Antibody Society traditionally held in San Diego in December. Membership is free for students, post-docs and employees of our corporate sponsors!

Most read from mAbs

by

The Antibody Society is pleased and proud to be affiliated with mAbs, a multi-disciplinary journal dedicated to advancing the art and science of antibody research and development. We hope you enjoy these brief summaries based on the abstracts of the most read papers published in recent issues. All the articles are open access; PDFs can be downloaded by following the links below.

Issue 10.5 (July 2018)

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. In this new review, Pereira et al. discuss the relevance of antibody core fucosylation to antibody-dependent cell-mediated cytotoxicity, and different strategies to produce afucosylated antibodies, and provide an update of afucosylated antibody drugs currently undergoing clinical trials, as well as those that have been approved.

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells. Sasso et al. report the results of their study of SINEUP technology applied to semi-stable production of monoclonal antibodies in HEK293E cells. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. The authors propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC. Figueroa et al. present a practical approach that uses limited pharmacokinetic (PK) and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. Their findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.

Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human, cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach. In this report, Betts et al. used population-pharmacokinetic (popPK) modeling to determine a single set of ‘typical’ popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.

Issue 10.4 (May/June 2018)

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. Bradbury et al. discuss results of their study, which analyzed 185 random hybridomas, in a large multicenter dataset, to determine the genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. Of the hybridomas evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. Their findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

Evaluation of analytical similarity between trastuzumab biosimilar CT-P6 and reference product using statistical analyses. In this report, Lee et al. evaluated analytical similarity of CT-P6, a biosimilar product of trastuzumab, with the reference products (EU-Herceptin® or US-Herceptin®) following risk-based statistical approaches recommended in a recent US Food and Drug Administration guideline for the risk-based statistical approaches recommended by the US Food and Drug Administration. Various quality attributes of trastuzumab were first ranked based on the clinical impact of each attribute and subsequently adjusted to one of three tiers (Tier 1, Tier 2 and Tier 3) considering the characteristics of the assay, the level of attribute present and the feasibility of statistical analysis. Analytical similarity assessment analyzed by the three tiers clearly demonstrated that CT-P6 exhibits highly similar structural and physicochemical properties, as well as functional activities, compared with the reference products.

Analytical and functional similarity of Amgen biosimilar ABP 215 to bevacizumab. Seo et al. report the results of their analytical similarity assessment, which was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

 

Like this post but not a member? We encourage you to join the Society to take advantage of the substantial benefits of membership, including discounts on fees for selected KNect365, CHI, and Hanson Wade meetings, discounted subscriptions to Society-affiliated journals PEDS and mAbs (special subscription rate of US $84 online only access for Antibody Society members)  and access to information in the Members Only section of the website. In particular, we encourage members to take advantage of the discount on registration for Antibody Engineering & Therapeutics, which is the annual meeting of The Antibody Society traditionally held in San Diego in December. Membership is free for students, post-docs and employees of our corporate sponsors!

Drop it and run

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Therapeutic antibodies have been successfully used for decades to treat various diseases. For antibodies targeting soluble antigens, however, a so-called “antibody buffering” effect, which can prolong the persistence of the target in the blood instead of clearing it, was observed. When a conventional IgG is injected into the body and binds to its corresponding antigen, the immune complexes are taken up into the cell where a certain amount of the antigen dissociates from the antibody in the endosomal compartments. The dissociated antigen is directed to the lysosomes for degradation, but the remaining amount of antigen (still bound to the IgG molecules) is recycled out of the cell by the neonatal Fc receptor (FcRn), and this can lead to an extension rather than a decrease of the antigen half-life in the bloodstream (1-5).

To overcome this buffering effect, antibodies with pH-dependent antigen binding characteristics were developed. These IgGs bind the soluble target molecules at physiological pH, but release antigen at the acidic pH in the endosomes. Antigen will then be directed into lysosomes for degradation and free antibodies will be recycled out of the cell, available for consecutive rounds of antigen binding and intracellular delivery. This method has been successfully applied to target different soluble antigens, demonstrating enhanced antigen clearance from the bloodstream compared to a conventional IgG with no pH-dependent antigen binding characteristics (6-9). Furthermore, to facilitate even more efficient antigen elimination, pH-dependent antibodies with additional modifications were generated. By increasing the antibody affinity for FcRn or FcyRIIb, soluble antigen bound to the engineered antibodies will enter the cell much more efficiently than by fluid-phase uptake. The combined effects of increased uptake and pH-dependent antigen dissociation resulted in a remarkable decrease of antigen levels following injection of engineered “sweeping” antibodies, opening possibilities for improved therapeutic applications in the future (10-13). We look forward to receiving further news about pH-dependent antibodies already in development (9, 14-15).

References:
1, Finkelman et al, J Immunol. 1993 Aug 1;151(3):1235-44.
2, O’Hear and Foote, Proc Natl Acad Sci U S A. 2005 Jan 4;102(1):40-4.
3, Phelan et al, J Immunol. 2008 Jan 1;180(1):44-8.
4, Davda and Hansen, MAbs. 2010 Sep-Oct;2(5):576-88. doi: 10.4161/mabs.2.5.12833.
5, Xiao et al, AAPS J. 2010 Dec;12(4):646-57. doi: 10.1208/s12248-010-9222-0.
6, Igawa et al, Nat Biotechnol. 2010 Nov;28(11):1203-7. doi: 10.1038/nbt.1691.
7, Chaparro-Riggers et al, J Biol Chem. 2012 Mar 30;287(14):11090-7. doi: 10.1074/jbc.M111.319764.
8, Devanaboyina et al, MAbs. 2013 Nov-Dec;5(6):851-9. doi: 10.4161/mabs.26389.
9, Fukuzawa et al, Sci Rep. 2017 Apr 24;7(1):1080. doi: 10.1038/s41598-017-01087-7.
10, Igawa et al, PLoS One. 2013 May 7;8(5):e63236. doi: 10.1371/journal.pone.0063236.
11, Iwayanagi et al, J Immunol. 2015 Oct 1;195(7):3198-205. doi: 10.4049/jimmunol.1401470.
12, Igawa et al, Immunol Rev. 2016 Mar;270(1):132-51. doi: 10.1111/imr.12392.
13, Yang et al, accepted manuscript, MAbs. 2017 Aug 8:0. doi: 10.1080/19420862.2017.1359455.
14, ALXN1210, https://clinicaltrials.gov/ct2/show/NCT02946463
15, SA237, https://clinicaltrials.gov/ct2/show/NCT02028884

Importance of isoelectric point (pI) of antibodies

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One important characteristic of monoclonal antibodies (mAbs) is their isoelectric point (pI), which essentially is the pH at which the antibody has no net electrical charge, and its value depends on the charged amino acids the antibody contains. If the pH of the surrounding environment is below the antibody’s pI, then the molecule carries a net positive charge, whereas the antibody will carry a net negative charge when the pH is above the pI.

When assessing pharmacokinetic (PK) properties of therapeutic antibodies not only the target-mediated drug disposition (TMDD) but also non-target-related mechanisms influence overall PK behavior and pI can be an important factor of the latter. Since the surface of most cells is negatively charged, antibodies need to be positively charged for efficient fluid-phase endocytosis (pinocytosis), therefore the environmental pH needs to be below the pI of the antibody. Therapeutic antibodies with pI values in the range of 8-9 are taken up adequately after administration since the physiological pH is 7.4, however, in some cases antibodies have a pI outside of this range or have been manipulated to achieve increased or decreased pI values (cationization or anionization, respectively). It is generally observed that increases in net positive charge of antibodies result in increased blood clearance and increased tissue retention with shorter half-life, whereas antibodies with lower pI generally have decreased tissue uptake and longer half-life (1-3), although observations can be conflicting regarding correlation between mAb clearance and pI (4). Even subtle manipulation such as molecular surface remodeling to disrupt positive patch regions can influence PK properties (5). In summary, pI of mAbs is known to have a substantial effect on PK behavior independent of recycling mediated by the neonatal Fc receptor, FcRn. Small changes of pI during the routine manufacturing of mAb charge variants, however, are not expected to result in dramatic changes and may not require extensive analyses (6). In any case, it is encouraged that pI values are reported in studies as an important factor influencing antibody behavior. Furthermore, since selecting the best candidates during early preclinical phases of product development can substantially decrease time and cost of development, it is of great importance to consider de-risk strategies and tools during drug discovery and development, including antibody variable region charge and antibody pI analyses (7-9). A new analytical platform has been recently described and validated on seven mAbs to rapidly assess and rank mAb charge variants during early stage development, which can be a useful screening technique during early stage development (10).

References:

1 Boswell et al, Bioconjug Chem. 2010 Dec 15;21(12):2153-63.
2 Igawa et al, Protein Eng Des Sel. 2010 May;23(5):385-92.
3, Li et al, MAbs. 2014;6(5):1255-64.
4, Hötzel et al, MAbs. 2012 Nov-Dec;4(6):753-60.
5, Datta-Mannan et al, MAbs. 2015;7(3):483-93.
6, Khawli et al, mAbs, 2010;(2)6:613-624.
7, Bumbaca Yadav et al, J Biol Chem. 2015 Dec 11;290(50):29732-41.
8, Dostalek et al, MAbs. 2017 May 2:1-11.
9, Jarasch et al, J Pharm Sci. 2015 Jun;104(6):1885-98.
10, Wagner-Rousset, J Chromatogr A. 2017 May 19;1498:147-154.