Biophysical properties of the clinical-stage antibody landscape.

Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.

Elimination of diaminopeptidase activity in Pichia pastoris for therapeutic protein production.

Yeast are important production platforms for the generation of recombinant proteins. Nonetheless, their use has been restricted in the production of therapeutic proteins due to differences in their glycosylation profile with that of higher eukaryotes. The yeast strain Pichia pastoris is an industrially important organism. Recent advances in the glycoengineering of this strain offer the potential to produce therapeutic glycoproteins with sialylated human-like N- and O-linked glycans. However, like higher eukaryotes, yeast also express numerous proteases, many of which are either localized to the secretory pathway or pass through it en route to their final destination. As a consequence, nondesirable proteolysis of some recombinant proteins may occur, with the specific cleavage being dependent on the class of protease involved. Dipeptidyl aminopeptidases (DPP) are a class of proteolytic enzymes which remove a two-amino acid peptide from the N-terminus of a protein. In P. pastoris, two such enzymes have been identified, Ste13p and Dap2p. In the current report, we demonstrate that while the knockout of STE13 alone may protect certain proteins from N-terminal clipping, other proteins may require the double knockout of both STE13 and DAP2. As such, this understanding of DPP activity enhances the utility of the P. pastoris expression system, thus facilitating the production of recombinant therapeutic proteins with their intact native sequences.

N-glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans.

N-linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95% derivatization was achieved in 30 min and the labeled glycans, when kept at -20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC-MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity.

High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies.

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.

Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.

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.

Affinity Maturation Enhances Antibody Specificity but Compromises Conformational Stability.

Monoclonal antibodies (mAbs) have recently emerged as one of the most promising classes of biotherapeutics. A potential advantage of B cell-derived mAbs as therapeutic agents is that they have been subjected to natural filtering mechanisms, which may enrich for B cell receptors (BCRs) with favorable biophysical properties. Here, we evaluated 400 human mAbs for polyreactivity, hydrophobicity, and thermal stability using high-throughput screening assays. Overall, mAbs derived from memory B cells and long-lived plasma cells (LLPCs) display reduced levels of polyreactivity, hydrophobicity, and thermal stability compared with naive B cell-derived mAbs. Somatic hypermutation (SHM) is inversely associated with all three biophysical properties, as well as BCR expression levels. Finally, the developability profiles of the human B cell-derived mAbs are comparable with those observed for clinical mAbs, suggesting their high therapeutic potential. The results provide insight into the biophysical consequences of affinity maturation and have implications for therapeutic antibody engineering and development.

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients.

Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.

Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies.

Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H2O2 for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.

Broad epitope coverage of a human in vitro antibody library.

Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

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.

Understanding ForteBio's Sensors for High-Throughput Kinetic and Epitope Screening for Purified Antibodies and Yeast Culture Supernatant.

Real-time and label-free antibody screening systems are becoming more popular because of the increasing output of purified antibodies and antibody supernatant from many antibody discovery platforms. However, the properties of the biosensor can greatly affect the kinetic and epitope binning results generated by these label-free screening systems. ForteBio human-specific ProA, anti-human IgG quantitation (AHQ), anti-human Fc capture (AHC) sensors, and custom biotinylated-anti-human Fc capture (b-AHFc) sensors were evaluated in terms of loading ability, regeneration, kinetic characterization, and epitope binning with both purified IgG and IgG supernatant. AHC sensors proved unreliable for kinetic or binning assays at times, whereas AHQ sensors showed poor loading and regeneration abilities. ProA sensors worked well with both purified IgG and IgG supernatant. However, the interaction between ProA sensors and the Fab region of the IgG with VH3 germline limited the application of ProA sensors, especially in the epitope binning experiment. In an attempt to generate a biosensor type that would be compatible with a variety of germlines and sample types, we found that the custom b-AHFc sensors appeared to be robust working with both purified IgG and IgG supernatant, with little evidence of sensor-related artifacts.

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.

An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies.

The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.

O-linked glycosylation analysis of recombinant human granulocyte colony-stimulating factor produced in glycoengineered Pichia pastoris by liquid chromatography and mass spectrometry.

Glycosylation is a major biochemical attribute of therapeutic proteins and detailed analyses including the structures and sites of such modifications are often required for product quality control and assurance. Using liquid chromatography and tandem mass spectrometry techniques, we analyzed the O-linked glycosylation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derived from glycoengineered Pichia pastoris with regard to its nature, structure, occupancy, and location. Peptide mappings using protease and chemical cleavages were performed to determine the specific O-linked glycosylation site used by Pichia-derived rhG-CSF. Our results demonstrated that Thr134, the equivalent O-linked glycosylation site found on endogenous human G-CSF, is the only site modified with a single mannose, allowing glycoengineered P. pastoris to be used as a viable production platform for therapeutic rhG-CSF.

Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry.

We developed a rapid method to analyze Fc glycosylation of Fc fusion proteins, especially those with mutated Fc hinge regions. Fc fusion proteins were digested with IdeS, an IgG specific protease with exosites for substrate recognition and cleavage. The resultant fragments were directly analyzed through liquid chromatography mass spectrometry. The structures and relative quantities of Fc glycans were deduced from their masses and intensities. The separated substrate recognition and cleavage property of IdeS makes this method applicable to a broad range of Fc fusion proteins having either standard or non-canonical hinge regions.

A cost-effective plate-based sample preparation for antibody N-glycan analysis.

During early cell line and process development of therapeutic antibodies, a cost-effective high-throughput approach to characterize the N-linked glycans is highly desired given that a large number of samples need to be analyzed. Using commercially available, low cost 96-well plates, we developed a practical procedure to prepare fluorescently labeled N-linked glycans for both qualitative and quantitative analysis by mass spectrometry (MS) and ultrahigh performance liquid chromatography (UPLC). Antibody samples were continuously denatured, reduced, and deglycosylated in a single 96-well hydrophobic membrane filter plate. Subsequently, released glycans were fluorescently labeled in a collection plate, and cleaned-up using a hydrophilic membrane filter plate. Carried out entirely in ready-to-use 96-well plates with simple buffer systems, this procedure requires less than 90min to finish. We applied the optimized procedure to examine the N-linked glycosylation of trastuzumab and were able to quantify ten major N-linked glycans. The results from different amounts of starting materials (10-200µg) were highly similar and showed the robustness of this procedure. Compared to other methods, this new procedure is simple to implement, economically more affordable, and could be very valuable for early screenings of antibody development.

Production of monoclonal antibodies by glycoengineered Pichia pastoris.

The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.