Analytical Hydrophobic Interaction Chromatography Research Articles

The role of intermolecular interactions on monoclonal antibody filtration through virus removal membranes.

The removal of viruses by filtration is a critical unit operation to ensure the overall safety of monoclonal antibody (mAb) products. Many mAbs show very low filtrate flux during virus removal filtration, although there are still significant uncertainties regarding both the mechanisms and antibody properties that determine the filtration behavior. Experiments were performed with three highly purified mAbs through 3 different commercial virus filters (Viresolve ® Pro, Viresolve® NFP, and Pegasus™ SV4) with different pore structures and chemistries. The flux decline observed during mAb filtration was largely reversible, even under conditions where the filtrate flux with the mAb was more than 100-fold smaller than the corresponding buffer flux. The extent of flux decline was highly correlated with the hydrodynamic diameter of the mAb as determined by dynamic light scattering. The mAb with the lowest filtrate flux for all 3 membranes showed the largest attractive intermolecular interactions and the greatest hydrophobicity, with the latter determined by binding to a butyl resin in an analytical hydrophobic interaction chromatography column. These results strongly suggest that the flux behavior is dominated by reversible self-association of the mAbs, providing important insights into the design of more effective virus filtration processes and in the early identification of problematic mAbs / solution conditions. This article is protected by copyright. All rights reserved.

Protein A and CaptureSelect FcXP Affinity Resins Possess the Capability of Differentiating Hole-hole Homodimer Isoforms.

Knobs-into-holes (KiH) technology has been widely used in asymmetric bispecific antibody (bsAb) construction to promote heavy chain heterodimerization. However, despite the great improvement of heterodimer formation by this strategy, homodimers (especially the hole-hole homodimer) can still be generated at low levels. Consequently, hole-hole homodimer is a common byproduct associated with the production of KiH bsAbs. In addition, previous studies showed that hole-hole homodimer exists as two different isoforms. As the major difference between these two isoforms lies in the Fc region, we speculated that Protein A media, which bind IgG Fc region with high affinity, and CaptureSelect FcXP, a CH3 domain-specific affinity resin, may provide a certain resolution between these two conformational isoforms. The objective of this study was to study the capability of Protein A and CaptureSelect FcXP affinity resins in differentiating hole-hole homodimer isoforms. The hole-hole homodimer was produced in CHO cells by expressing the hole half-antibody. The homodimer, along with the half-antibody was initially captured by Protein A chromatography and was then further purified by size-exclusion chromatography (SEC), which separated the homodimer from the unpaired half-antibody. The purified hole-hole homodimer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical hydrophobic interaction chromatography (HIC). The purified hole-hole homodimer was separately processed by columns packed with Protein A and CaptureSelect FcXP resins. The purified hole-hole homodimer was also analyzed by Protein A-high-performance liquid chromatography (HPLC). SDS-PAGE analysis and analytical HIC study confirmed that hole-hole homodimer exists as two conformational isoforms. When the hole-hole homodimer was processed by Protein A and CaptureSelect FcXP chromatography, the elution profiles contained two peaks, indicating that both affinity resins possess the capability of differentiating hole-hole homodimer isoforms. Our data suggest that Protein A and CaptureSelect FcXP affinity resins both possess the capability of differentiating hole-hole homodimer isoforms and, therefore, can be used for monitoring isoform conversion under various conditions.

Evaluating Nanoparticle Hydrophobicity Using Analytical Membrane Hydrophobic Interaction Chromatography.

Nanoparticle hydrophobicity is a key factor controlling the stability, adhesion, and transport of nanoparticle suspensions. Although a number of approaches have been presented for evaluating nanoparticle hydrophobicity, these methods are difficult to apply to larger nanoparticles and viruses (>100 nm in size) that are of increasing importance in drug delivery and gene therapy. This study investigated the use of a new analytical hydrophobic interaction chromatography method employing a 5.0 μm pore size polyvinylidene fluoride membrane as the stationary-phase in membrane hydrophobic interaction chromatography (MHIC). Experimental data obtained using a series of model proteins were in good agreement with literature values for the hydrophobicity (both experimental and computational). MHIC was then used to evaluate the hydrophobicity of a variety of nanoparticles, including a live attenuated viral vaccine, both in water and in the presence of different surfactants. This new method can be implemented on any liquid chromatography system, run times are typically <20 min, and the experiments avoid the use of organic solvents that could alter the structure of many biological nanoparticles.

Online coupling of analytical hydrophobic interaction chromatography with native mass spectrometry for the characterization of monoclonal antibodies and related products

Native mass spectrometry (native MS) has seen tremendous development and an increase in application over the past decade for the study of proteins and protein complexes. Although conventionally performed using a static nanospray emitter in an offline fashion, native MS has been increasingly applied in hyphenated methods, where a wide variety of separation techniques are directly coupled to online native MS detection. Those new developments have greatly expanded the utility of native MS in protein biopharmaceutical characterization. Analytical hydrophobic interaction chromatography (HIC) method, although frequently used for the characterization of monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), has rarely been explored for online coupling with native MS. This is largely due to the high salt concentrations used in HIC analysis that are not compatible with direct MS detection. In this study, we overcame this challenge via an innovative makeup and splitting flow design and successfully achieved online coupling of analytical HIC separation with native MS detection. The development and experimental setup of this HIC-MS method is outlined in detail to elucidate how this design could tackle the high salt concentrations used in HIC separation and ultimately achieve both good chromatographic resolution and MS data quality. Subsequently, the utility of this HIC-MS method was demonstrated in three different applications, where a mAb mixture, mAb molecular variants resulting from PTMs, and a Cys-based ADC mimic were all readily characterized in detail. Unlike previously reported HIC-MS methods, this newly developed method utilizes an analytical scale HIC column with conventional ligand so that the achieved separation profile is highly comparable to those obtained by a standard HIC-UV method. As a result, this HIC-MS method not only provides an alternative approach for in-depth characterization of mAbs and related products during their development but could also be readily applied to assist peak assignment and identity elucidation for the HIC-UV method used in quality control.

Monitoring removal of hole-hole homodimer by analytical hydrophobic interaction chromatography in purifying a bispecific antibody

Despite the use of knobs-into-holes (KiH) strategy to promote heterodimerization in recombinant production of bispecific antibody (bsAb), homodimer (especially the hole-hole homodimer) can still be generated in small amount. This by-product needs to be removed by downstream process. However, as homodimer and the target bsAb are usually very close in size, these two species may not be readily differentiated using size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Thus, method other than SEC-HPLC needs to be developed to monitor removal of this by-product. Here, through a case study we demonstrate that analytical hydrophobic interaction chromatography (HIC) is a powerful tool for quantitatively monitoring removal of hole-hole homodimer in bsAb purification.

Purification of TNFR-Fc produced in recombinant CHO cells: Characterization of product-related impurities

Because of its large size and structural complexity, preparations of the cysteine-rich tumor necrosis factor receptor-Fc (TNFR-Fc) that are produced using recombinant CHO cells contain many undesirable impurities. In this study, to purify TNFR-Fc, cell culture supernatant was first clarified using a pre-filter and sterilization filter and then subjected to a series of purification steps consisting of protein A affinity column chromatography, anion exchange chromatography, and hydrophobic interaction chromatography (HIC). To characterize the presence of TNFR-Fc-related impurities after the HIC step, the HIC eluates were further fractionated using analytical HIC and then separated by size exclusion chromatography (SEC). Several product-related impurities were detected during SEC, including low molecular weight (LMW) species, high molecular weight aggregates, and species with a size equivalent to authentic TNFR-Fc. N-terminal sequence analysis of the LMW species indicated that N-terminal amino acids had been partially deleted from the protein sequence at amino acid positions 1–185 or 1–223. Peptide mapping analysis followed by quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and MS/MS indicated that the species that was equivalent in size to authentic TNFR-Fc contained scrambled disulfide bonds linked as Cys98-Cys115 or Cys104-Cys112. These product-related impurities, which led to a marked reduction in TNF-α neutralizing activity during cytotoxicity neutralization assays in L929 cells, should be removed from the final product during purification.

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.

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule

Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.

Large-scale preparation of thrombin from human plasma

Thrombin was prepared from human blood plasma (batch size 1200 L). First, prothrombin was isolated by the following separation techniques: cryoprecipitation, ion-exchange chromatography (diethyl aminoethyl, DEAE-IEX), heparin affinity chromatography, a second DEAE-IEX step, and immobilized metal-affinity chromatography (IMAC). Prothrombin was then activated to thrombin, which was purified by hydrophobic interaction chromatography (HIC) and concentrated by ultrafiltration. This process is cost-effective because a waste fraction can be used from one of the steps (heparin affinity chromatography) in the commercial production of plasma-derived Factor IX (FIX). The final thrombin preparation had a purity of approximately 75% (specific activity approximately 2400 IU/mg protein), which is sufficient for its intended purpose in a fibrin glue. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Factor X (FX) activity analysis, and analytical HIC were also used to characterize the thrombin. Three substantially different techniques were used to reduce any viral activity, namely: solvent/detergent (S/D) treatment, pasteurization, and virus filtration (nanofiltration). The manufacturing process presented here would be suitable for large-scale production of thrombin with a high degree of virus safety.

An automated in vitro protein folding screen applied to a human dynactin subunit.

The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies). Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives. However, it is difficult to assess the success of such a screen if no biological assay is available. We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy. The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E. coli. The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses.

Optimization of preparative hydrophobic interaction chromatographic purification methods

The chromatographic behavior of five proteins on hydrophobic interaction matrices having six different ligand arms was investigated using gradient elution with ammonium sulfate and ammonium acetate buffers at two pH values. The nature of the mobile phase and/or the ligand chain arm of the matrix was found to have substantial effect on the resolution, retention, and selectivity. Ovalbumin was moderately or highly retained with ammonium sulfate on all columns; however, with ammonium acetate, ovalbumin was not retained on SynChropak Hydroxypropyl and Propyl columns. Chromatographic conditions developed for analytical hydrophobic interaction chromatography columns containing 6.5-μm packings were adapted to preparative columns packed with 30-μm SynChroprep packings for the separation of serum components. Dynamic load capacities were 4–13 mg of ovalbumin per ml of column volume.