High throughput screening for rapid and reliable prediction of monovalent antibody binding behavior in flowthrough mode.

Flowthrough (FT) anion exchange (AEX) chromatography is a widely used polishing step for the purification of monoclonal antibody (mAb) formats. To accelerate downstream process development, high throughput screening (HTS) tools have proven useful. In this study, the binding behavior of six monovalent mAbs (mvAbs) was investigated by HTS in batch binding mode on different AEX and mixed-mode resins at process-relevant pH and NaCl concentrations. The HTS entailed the evaluation of mvAb partition coefficients (Kp ) and visualization of results in surface-response models. Interestingly, the HTS data grouped the mvAbs into either a strong-binding group or a weak-binding/FT group independent of theoretical Isoelectric point. Mapping the charged and hydrophobic patches by in silico protein surface property analyses revealed that the distribution of patches play a major role in predicting FT behavior. Importantly, the conditions identified by HTS were successfully verified by 1 mL on-column experiments. Finally, employing the optimal FT conditions (7-9 mS/cm and pH 7.0) at a mini-pilot scale (CV = 259 mL) resulted in 99% yield and a 21-23-fold reduction of host cell protein to <100 ppm, depending on the varying host cell protein (HCP) levels in the load. This work opens the possibility of using HTS in FT mode to accelerate downstream process development for mvAb candidates in early research.

Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization.

PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation. METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations. RESULTS: We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively. CONCLUSIONS: We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.

Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

The immunological effects of oral polio vaccine provided with BCG vaccine at birth: a randomised trial.

BACKGROUND: Vaccines may have non-specific effects. An observational study from Guinea-Bissau suggested that oral polio vaccine at birth (OPV0) provided with Bacillus Calmette-Guérin (BCG) vaccine was associated with down-regulation of the immune response to BCG vaccine 6 weeks later. Based on the previous finding, we wanted to test our a priori hypothesis that OPV would dampen the immune response to BCG, and secondarily to test immune responses to other antigens. METHODS: The study was conducted at the Bandim Health Project in Guinea-Bissau in 2009-2010. Infants were randomised to OPV0+BCG versus BCG alone at birth, and subsequently randomised to have a blood sample taken at 2, 4 or 6 weeks post-randomisation. Excreted levels of cytokines (IL-2, IL-5, IL-10, TNF-α and IFN-γ) were measured from whole blood in vitro stimulations with a panel of recall vaccine antigens (BCG, PPD, OPV), mitogen (PHA) or innate agonists (LPS, Pam3cys, PolyI:C). Additionally, we measured the local reaction to BCG, white blood cell distribution, C-reactive protein (CRP) and retinol-binding protein (RBP). Cytokine production was analysed as the prevalence ratios of responders above the median. RESULTS: Blood samples from 430 infants (209 OPV0+BCG; 221 BCG alone) were analysed. There were no strong differences in effects 2, 4 and 6 weeks post-randomisation and subsequent analyses were performed on the pooled data. As hypothesised, receiving OPV0+BCG versus BCG alone was associated with significantly lower prevalence of IFN-γ responses to PPD (prevalence ratio (PR): 0.84 (0.72-0.98)) and reduced IL-5 to PPD (PR: 0.78 (0.64-0.96)). No effects were observed for CPR, RBP, white blood cell distribution, or BCG scar prevalence. CONCLUSION: The results corroborate that OPV attenuates the immune response to co-administered BCG at birth.

Model-based process development for the purification of a modified human growth hormone using multimodal chromatography.

This study demonstrates how the multimodal Capto adhere resin can be used in concert with calcium chloride or arginine hydrochloride as mobile phase modifiers to create a highly selective purification process for a modified human growth hormone. Importantly, these processes are shown to result in significant clearance of product related aggregates and host cell proteins. Furthermore, the steric mass action model is shown to be capable of accurately describing the chromatographic process and the aggregate removal. Finally, justification of the selected operating ranges is evaluated using the model together with Latin hypercube sampling. The results in this article establish the utility of multimodal chromatography when used with appropriate mobile phase modifiers for the downstream bioprocessing of a modified human growth hormone and offer new approaches for bioprocess verification.

Methods development in multimodal chromatography with mobile phase modifiers using the steric mass action model.

The ability to predict downstream protein purification processes is of great value in the biopharmaceutical industry; saving time, cost and resources. While many complex models exist, the appropriate use of simple models can be a useful tool for rapidly designing and optimizing processes as well as for risk analysis and establishing parameter ranges. In this study, the steric mass action isotherm is success-fully employed to predict the chromatographic behavior of a multimodal anionic Capto adhere systemin the presence of various mobile phase modifiers. An experimental protocol consisting of only a few column experiments is shown to be sufficient to establish the model. Proof of concept is carried out using human insulin and bovine serum albumin which have varying degrees of hydrophobicity, charge and size. Finally, the model predictions are verified under various experimental conditions and the unique selectivity of this multimodal system is explored and compared with traditional anion exchange resins.The simple model approach described here represents a rapid and useful method for model based process development of multimodal chromatography.