Early developability screen of therapeutic antibody candidates using Taylor dispersion analysis and UV area imaging detection

Therapeutic antibodies represent one of the fastest growing segments in the pharmaceutical market. They are used in a broad range of disease fields, such as autoimmune diseases, cancer, inflammation and infectious diseases. The growth of the segment has necessitated development of new analytical platforms for faster and better antibody selection and characterization. Early quality control and risk assessment of biophysical parameters help prevent failure in later stages of antibody development, and thus can reduce costs and save time. Critical parameters such as aggregation, conformational stability, colloidal stability and hydrophilicity, are measured during the early phase of antibody generation and guide the selection process of the best lead candidates in terms of technical developability. We report on the use of a novel instrument (ActiPix/Viscosizer) for measuring both the hydrodynamic radius and the absolute viscosity of antibodies based on Taylor dispersion analysis and UV area imaging. The looped microcapillary-based method combines low sample consumption, fast throughput and high precision compared to other conventional methods. From a random panel of 130 antibodies in the early selection process, we identified some with large hydrodynamic radius outside the normal distribution and others with non-Gaussian Taylor dispersion profiles. The antibodies with such abnormal properties were confirmed later in the selection process to show poor developability profiles. Moreover, combining these results with those of the viscosity measurements at high antibody concentrations allows screening, with limited amounts of materials, candidates with potential issues in pre-formulation development.     

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.     

Structure,heterogeneity and developability assessment of therapeutic antibodies

Increasing attention has been paid to developability assessment with the understanding that thorough evaluation of monoclonal antibody lead candidates at an early stage can avoid delays during late-stage development. The concept of developability is based on the knowledge gained from the successful development of approximately 80 marketed antibody and Fc-fusion protein drug products and from the lessons learned from many failed development programs over the last three decades. Here, we reviewed antibody quality attributes that are critical to development and traditional and state-of-the-art analytical methods to monitor those attributes. Based on our collective experiences, a practical workflow is proposed as a best practice for developability assessment including in silico evaluation, extended characterization and forced degradation using appropriate analytical methods that allow characterization with limited material consumption and fast turnaround time.     

A novel screening method to assess developability of antibody-like molecules

Monoclonal antibodies and antibody-like molecules represent a fast-growing class of bio-therapeutics that has rapidly transformed patient care in a variety of disease indications. The discovery of antibodies that bind to particular targets with high affinity is now a routine exercise and a variety of in vitro and in vivo techniques are available for this purpose. However, it is still challenging to identify antibodies that, in addition to having the desired biological effect, also express well, remain soluble at different pH levels, remain stable at high concentrations, can withstand high shear stress, and have minimal non-specific interactions. Many promising antibody programs have ultimately failed in development due to the problems associated with one of these factors. Here, we present a simple high-performance liquid chromatography (HPLC)-based screening method to assess these developability factors earlier in discovery process. This method is robust and requires only microgram quantities of proteins. Briefly, we show that for antibodies injected on a commercially available pre-packed Zenix HPLC column, the retention times are inversely related to their colloidal stability with antibodies prone to precipitation or aggregation retained longer on the column with broader peaks. By simply varying the salt content of running buffer, we were also able to estimate the nature of interactions between the antibodies and the column. We believe this approach should generally be applicable to assessment of the developability of other classes of bio-therapeutic molecules, and that the addition of this simple tool early in the discovery process will lead to selection of molecules with improved developability characteristics.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

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.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH2 domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.     

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.     

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational,structure-guided Fv engineering

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.     

Comparison of surface plasmon resonance binding curves for characterization of protein interactions and analysis of screening data

Label-free technologies, such as surface plasmon resonance, are typically used for characterization of protein interactions and in screening for selection of antibodies or small molecules with preferred binding properties. In characterization, complete binding curves are normally fitted to defined interaction models to provide affinity and rate constants, whereas report points indicative of binding and stability of binding are often used for analysis of screening data. As an alternative to these procedures, here we describe how the analysis, in certain cases, can be simplified by comparison with upper and lower limit binding curves that represent expected or wanted binding profiles. The use of such profiles is applied to the analysis of kinetically complex IgG–Fc receptor interactions and for selection of antibody candidates. The comparison procedure described may be particularly useful in batch-to-batch comparisons and in comparability and biosimilar studies of biotherapeutic medicines. In screening, more informed selections may become possible as entire binding profiles and not a few report points are used in the analysis and as each new sample is directly compared with a predefined outcome.     

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self-interaction and polyspecificity compared to non-phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non-phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation-prone natures of antibodies which should be avoided in early development phases.     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH₂ domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.Key words: monoclonal antibody, glycosylation, stability, liquid chromatography-mass spectroscopy, Fourier transform infrared, fluorescence spectroscopy, size-exclusion chromatography, differential scanning calorimetry     

Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored.     

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.     

Commentary: Why Pharmaceutical Scientists in Early Drug Discovery Are Critical for Influencing the Design and Selection of Optimal Drug Candidates

This commentary reflects the collective view of pharmaceutical scientists from four different organizations with extensive experience in the field of drug discovery support. Herein, engaging discussion is presented on the current and future approaches for the selection of the most optimal and developable drug candidates. Over the past two decades, developability assessment programs have been implemented with the intention of improving physicochemical and metabolic properties. However, the complexity of both new drug targets and non-traditional drug candidates provides continuing challenges for developing formulations for optimal drug delivery. The need for more enabled technologies to deliver drug candidates has necessitated an even more active role for pharmaceutical scientists to influence many key molecular parameters during compound optimization and selection. This enhanced role begins at the early in vitro screening stages, where key learnings regarding the interplay of molecular structure and pharmaceutical property relationships can be derived. Performance of the drug candidates in formulations intended to support key in vivo studies provides important information on chemotype-formulation compatibility relationships. Structure modifications to support the selection of the solid form are also important to consider, and predictive in silico models are being rapidly developed in this area. Ultimately, the role of pharmaceutical scientists in drug discovery now extends beyond rapid solubility screening, early form assessment, and data delivery. This multidisciplinary role has evolved to include the practice of proactively taking part in the molecular design to better align solid form and formulation requirements to enhance developability potential.     

Multiscale Coarse-Grained Approach to Investigate Self-Association of Antibodies

Self-association of therapeutic monoclonal antibodies (mabs) are thought to modulate the undesirably high viscosity observed in their concentrated solutions. Computational prediction of such a self-association behavior is advantageous early during mab drug candidate selection when material availability is limited. Here, we present a coarse-grained (CG) simulation method that enables microsecond molecular dynamics simulations of full-length antibodies at high concentrations. The proposed approach differs from others in two ways: first, charges are assigned to CG beads in an effort to reproduce molecular multipole moments and charge asymmetry of full-length antibodies instead of only localized charges. This leads to great improvements in the agreement between CG and all-atom electrostatic fields. Second, the distinctive hydrophobic character of each antibody is incorporated through empirical adjustments to the short-range van der Waals terms dictated by cosolvent all-atom molecular dynamics simulations of antibody variable regions. CG simulations performed on a set of 15 different mabs reveal that diffusion coefficients in crowded environments are markedly impacted by intermolecular interactions. Diffusion coefficients computed from the simulations are in correlation with experimentally measured observables, including viscosities at a high concentration. Further, we show that the evaluation of electrostatic and hydrophobic characters of the mabs is useful in predicting the nonuniform effect of salt on the viscosity of mab solutions. This CG modeling approach is particularly applicable as a material-free screening tool for selecting antibody candidates with desirable viscosity properties.     

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.     

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.