Heather Lynaugh

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.

Biophysical properties of the clinical-stage antibody landscape

Significance In addition to binding to a desired target molecule, all antibody drugs must also meet a set of criteria regarding the feasibility of their manufacture, stability in storage, and absence of off-target stickiness. This suite of characteristics is often termed “developability.” We present here a comprehensive analysis of these properties for essentially the full set of antibody drugs that have been tested in phase-2 or -3 clinical trials, or are approved by the FDA. Surprisingly, many of the drugs or candidates in this set exhibit properties that indicate significant developability risks; however, the number of such red warning flags decreases with advancement toward approval. This reference dataset should help prioritize future drug candidates for development. 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.

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.

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.

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

ABSTRACT 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.

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.

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

ABSTRACT 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.

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.