In vitro and in silico assessment of the developability of a designed monoclonal antibody library

Despite major advances in antibody discovery technologies, the successful development of monoclonal antibodies (mAbs) into effective therapeutic and diagnostic agents can often be impeded by developability liabilities, such as poor expression, low solubility, high viscosity and aggregation. Therefore, strategies to predict at the early phases of antibody development the risk of late-stage failure of antibody candidates are highly valuable. In this work, we employ the in silico solubility predictor CamSol to design a library of 17 variants of a humanized mAb predicted to span a broad range of solubility values, and we examine their developability potential with a battery of commonly used in vitro and in silico assays. Our results demonstrate the ability of CamSol to rationally enhance mAb developability, and provide a quantitative comparison of in vitro developability measurements with each other and with more resource-intensive solubility measurements, as well as with in silico predictors that offer a potentially faster and cheaper alternative. We observed a strong correlation between predicted and experimentally determined solubility values, as well as with measurements obtained using a panel of in vitro developability assays that probe non-specific interactions. These results indicate that computational methods have the potential to reduce or eliminate the need of carrying out laborious in vitro quality controls for large numbers of lead candidates. Overall, our study provides support to the emerging view that the implementation of in silico tools in antibody discovery campaigns can ensure rapid and early selection of antibodies with optimal developability potential.     

High throughput cross-interaction measures for human IgG1 antibodies correlate with clearance rates in mice

Although improvements in technology for the isolation of potential therapeutic antibodies have made the process increasingly predictable, the development of biologically active monoclonal antibodies (mAbs) into drugs can often be impeded by developability issues such as poor expression, solubility, and promiscuous cross-reactivity. Establishing early stage developability screening assays capable of predicting late stage behavior is therefore of high value to minimize development risks. Toward this goal, we selected a panel of 16 monoclonal antibodies (mAbs) representing different developability profiles, in terms of self- and cross-interaction propensity, and examined their downstream behavior from expression titer to accelerated stability and pharmacokinetics in mice. Clearance rates showed significant rank-order correlations to 2 cross-interaction related assays, with the closest correlation to a non-specificity assay on the surface of yeast. Additionally, 2 self-association assays correlated with each other but not to mouse clearance rate. This case study suggests that combining assays capable of high throughput screening of self- and cross-interaction early in the discovery stage could significantly lower downstream development risks.     

Target-independent variable region mediated effects on antibody clearance can be FcRn independent

The importance of the neonatal Fc receptor (FcRn) in extending the serum half-life of monoclonal antibodies (mAbs) is well demonstrated, and has led to the development of multiple engineering approaches designed to alter Fc interactions with FcRn. Recent reports have additionally highlighted the effect of nonspecific interactions on antibody pharmacokinetics (PK), suggesting an FcRn-independent mechanism for mAb clearance. In this report we examine a case study of 2 anti-interleukin-12/23 antibodies, ustekinumab and briakinumab, which share the same target and Fc, but differ in variable region sequences. Ustekinumab displayed near baseline signal in a wide range of early stage developability assays for undesirable protein/protein interactions, while briakinumab showed significant propensity for self- and cross-interactions. This phenotypic difference correlates with faster clearance rates for briakinumab in both human FcRn transgenic and FcRn knockout mice. These findings support a dominant contribution for FcRn-independent clearance for antibodies with high nonspecificity, and highlight a key role for early stage developability screening to eliminate clones with such high nonspecific disposition PK.     

A simple assay for detection of small-molecule redox activity

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts.     

Aggregation risk prediction for antibodies and its application to biotherapeutic development

Aggregation is a common problem affecting biopharmaceutical development that can have a significant effect on the quality of the product, as well as the safety to patients, particularly because of the increased risk of immune reactions. Here, we describe a new high-throughput screening algorithm developed to classify antibody molecules based on their propensity to aggregate. The tool, constructed and validated on experimental aggregation data for over 500 antibodies, is able to discern molecules with a high aggregation propensity as defined by experimental criteria relevant to bioprocessing and manufacturing of these molecules. Furthermore, we show how this tool can be combined with other computational approaches during early drug development to select molecules with reduced risk of aggregation and optimal developability properties.     

Aggregation risk prediction for antibodies and its application to biotherapeutic development

Aggregation is a common problem affecting biopharmaceutical development that can have a significant effect on the quality of the product, as well as the safety to patients, particularly because of the increased risk of immune reactions. Here, we describe a new high-throughput screening algorithm developed to classify antibody molecules based on their propensity to aggregate. The tool, constructed and validated on experimental aggregation data for over 500 antibodies, is able to discern molecules with a high aggregation propensity as defined by experimental criteria relevant to bioprocessing and manufacturing of these molecules. Furthermore, we show how this tool can be combined with other computational approaches during early drug development to select molecules with reduced risk of aggregation and optimal developability properties.     

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.     

Establishment of the platform for reverse chemical genetics targeting novel protein-protein interactions

In the "drug discovery" era, protein-protein interaction modules are becoming the most exciting group of targets for study. Although combinatorial libraries and active natural products are rapidly and systemically being equipped by both for-profit and not-for-profit organizations, complete drug-screening systems have not been achieved. There is a growing need for the establishment of drug discovery assays for highly effective utilization of the collected small molecules on a large scale. To generate drug-screening systems, we plan to identify novel protein-protein interactions that may participate in human diseases. The interactions have been identified by MS/MS analysis following immunoprecipitation using antibodies prepared from our cDNA projects. The intracellular pathway involving the identified interaction is computationally constructed, which then clarifies its relationship to the candidate disease. The development of reverse chemical genetics based on such information should help us to realize a significant increment in the number of drug discovery assays available for use. In this article, I describe our strategy for drug discovery and then introduce the applicability of fluorescence intensity distribution analysis (FIDA) and the expression-ready constructs called "ORF trap clones" to reverse chemical genetics.     

The making of bispecific antibodies

During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The ‘zoo’ of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term ‘developability’. In addition, different ‘target product profiles’, i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not ‘one best format’ for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.     

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies

Contemporary in vivo and in vitro discovery platform technologies greatly increase the odds of identifying high-affinity monoclonal antibodies (mAbs) towards essentially any desired biologically relevant epitope. Lagging discovery throughput is the ability to select for highly developable mAbs with drug-like properties early in the process. Upstream consideration of developability metrics should reduce the frequency of failures in later development stages. As the field moves towards incorporating biophysical screening assays in parallel to discovery processes, similar approaches should also be used to ensure robust chemical stability. Optimization of chemical stability in the early stages of discovery has the potential to reduce complications in formulation development and improve the potential for successful liquid formulations. However, at present, our knowledge of the chemical stability characteristics of clinical-stage therapeutic mAbs is fragmented and lacks comprehensive comparative assessment. To address this knowledge gap, we produced 131 mAbs with amino acid sequences corresponding to the variable regions of clinical-stage mAbs, subjected these to low and high pH stresses and identified the resulting modifications at amino acid-level resolution via tryptic peptide mapping. Among this large set of mAbs, relatively high frequencies of asparagine deamidation events were observed in CDRs H2 and L1, while CDRs H3, H2 and L1 contained relatively high frequencies of instances of aspartate isomerization.     

Predictive tools for stabilization of therapeutic proteins

Monoclonal antibodies represent the fastest growing class of pharmaceuticals. A major problem, however, is that the proteins are susceptible to aggregation at the high concentration commonly used during manufacturing and storage. Our recent publication describes a technology based on molecular simulations to identify aggregation-prone regions of proteins in silico. The technology, called spatial aggregation propensity (SAP), identifies hot-spots for aggregation based on the dynamic exposure of spatially-adjacent hydrophobic amino acids. Monoclonal antibodies (mAbs) in which patches with high-SAP scores are changed to patches with significantly reduced SAP scores via a single mutation are more stable than wild type, thus validating the SAP method for mapping aggregation-prone regions on proteins. We propose that the SAP technology will be useful for protein stabilization, and as a screening tool to bridge discovery and development of protein-based therapeutics by a rational assessment of the developability of candidate protein drugs.     

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.     

Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 microm i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.     

High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.     

A survey of the year 2002 commercial optical biosensor literature

We have compiled 819 articles published in the year 2002 that involved commercial optical biosensor technology. The literature demonstrates that the technology's application continues to increase as biosensors are contributing to diverse scientific fields and are used to examine interactions ranging in size from small molecules to whole cells. Also, the variety of available commercial biosensor platforms is increasing and the expertise of users is improving. In this review, we use the literature to focus on the basic types of biosensor experiments, including kinetics, equilibrium analysis, solution competition, active concentration determination and screening. In addition, using examples of particularly well-performed analyses, we illustrate the high information content available in the primary response data and emphasize the impact of including figures in publications to support the results of biosensor analyses.     

Advances in the use of biotinylated diaminopyridine (BAP) as a versatile fluorescent tag for oligosaccharides

We recently described a novel fluorescent compound, 2-amino,6-amidobiotinyl-pyridine(BAP), that allows the tagging of oligosaccharides, their fractionationby reversed-phase HPLC with picomole scale detection, and theformation of functional neoglycoprotein equivalents with (strept)avidin for the detection of receptors and the generation ofmonospedflc antibodies (Rothenberg etaL, Proc. Natl Acad. SetUSA, 90,11939-11943,1993). Here, we describe the enhancementof this approach by the following, (i) A simple one-step purificationof BAP from its synthetic precursors and other reactants. (ii)Development of HPLC sizing column methods to quickly purifyBAP-coupled oligosaccharides away from free BAP and other reactants.(iii) Development of anion-exchange and amine-adsorption HPLCprocedures for the fractionation of BAP-oligo-saccharide adductsby charge and size, respectively, (iv) Investigation of theaffinity of BAP-oligosaccharides for (strept)avidin, confirmingthe formation of stable complexes, (v) The use of BAP for sensitivemonosaccharide compositional analysis of glycoproteins. (vi)Formation of stable BAP adducts without reduction and its implicationsfor the mechanism of adduct formation. These advances make availablea multitude of techniques for the fractionation of BAP-coupledoligosaccharides based on several different physical parameters.Distinct species of BAP-coupled oligosacharides can be isolatedand subjected to detailed structural analysis. Such definedmolecules form stable complexes with streptavidin that are effectivelyneo-glycoproteins, which can be used in a variety of biologicalapplications. Notably, all of these approaches require relativelyinexpensive materials and conventional equipment available Inmost laboratories. analysis biotin fluorescence neoglycoconjugates     

Affinity-purification coupled to mass spectrometry: basic principles and strategies

Identifying the interactions established by a protein of interest can be a critical step in understanding its function. This is especially true when an unknown protein of interest is demonstrated to physically interact with proteins of known function. While many techniques have been developed to characterize protein-protein interactions, one strategy that has gained considerable momentum over the past decade for identification and quantification of protein-protein interactions, is affinity-purification followed by mass spectrometry (AP-MS). Here, we briefly review the basic principles used in affinity-purification coupled to mass spectrometry, with an emphasis on tools (both biochemical and computational), which enable the discovery and reporting of high quality protein-protein interactions.     

Enzymatic and Radioimmunoassay Procedures Combined with Electrophoresis and HPLC for the Recovery and Characterization of Substance P in Human Brain

Immunoreactive substance P was recovered from human brain (hypothalamus and substantia nigra) by acetic acid extraction, ion exchange chromatography (SP-Sephadex), molecular sieving (Sephadex C-50) and column electrophoresis in agarose suspension. The chemical nature of the active material was further studied with various biochemical techniques including agarose suspension electrophoresis, HPLC and different kinds of enzyme radioimmunoassays. By combining these techniques it was possible to confirm structure identity between the recovered active component and substance P previously isolated from bovine brain. Thus, the major activity reacting with the substance P antibodies was indistinguishable from the synthetic bovine analogue in all chromatographic systems including analytical electrophoresis at different pH:s and HPLC. Furthermore, digestion of the active material with post-proline cleaving enzyme and trypsin yielded fragments identical with those expected from the bovine peptide as confirmed by specific radioimmunoassays in conjuction with electrophoresis or HPLC. The result also indicates the usefulness of the present procedures for identifying peptides structures available only in minute amounts.     

Use of a new pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds

Good results have been obtained with use of the new bonded chiral stationary phase Whelk-O 1 in analytical and preparative subcritical fluid chromatography. A wide variety of enantiomeric pairs of compounds with different functional groups that are of pharmaceutical and biological interest have been resolved. This Pirkle-concept CSP appears to be more rugged than cellulosic phases (e.g., Chiralcel) with regards to solvents and pressure. In comparing the usefulness of the column for SFC versus HPLC chiral analysis, we have observed a clear superiority of SFC in terms of higher speed and efficiency of analysis, and faster method development. This is consistent with our experience with Chiralcel CSPs. With the Whelk-O 1 we have shown that the effects of temperature and modifier on SFC separations are similar to what has been reported for most other CSPs. We also observed a unique selectivity advantage of SFC for verapamil. We had good success with using a 1-in. diameter column packed with Whelk-O 1 to perform preparative SFC separations of a number of enantiomeric mixtures. The advantages of preparative SFC over preparative HPLC will be discussed. The feasibility of preparative SFC is dependent on how well we meet the practical challenges such as sample introduction issues, special hardware requirements due to the high pressure, and fraction collection issues. © 1994 Wiley-Liss, Inc.     

A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples

Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as approximately 100 pg (approximately 10(-13) mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.