High-Concentration Antibody Formulations Research Articles

Leveraging high-throughput analytics and automation to rapidly develop high-concentration mAb formulations: integrated excipient compatibility and viscosity screening

Abstract Background: Formulation screening is essential to experimentally balance stability and viscosity in high-concentration mAb formulations. We developed a high-throughput approach with automated sample preparation and analytical workflows to enable the integrated assessment of excipient compatibility and viscosity of mAb formulations. Methods: Ninety-six formulations of a trastuzumab biosimilar were screened by combining 8 types of excipient modifiers with 4 types of buffers across a pH range of 4.5 to 7.5. Key stability risks, including high molecular weight (HMW) aggregation and fragmentation, were thoroughly assessed along with viscosity at high concentrations. Additionally, several biophysical parameters were evaluated for their ability to predict stability or viscosity outcomes. Multiple linear regression was applied to fit the data and identify key factors. Results: The optimal pH range for the trastuzumab biosimilar was found to be 5.0 to 6.5, based on opposing pH dependencies for stability and viscosity. Buffer type had a minor effect on viscosity and fragmentation but played a significant role in influencing HMW aggregates, with Na-acetate and histidine-HCl being the best candidates. The impact of excipient modifiers on viscosity, HMW, and fragmentation depended on both pH and buffer type, showing strong interactions among factors. Arginine-HCl and lysine-HCl effectively lowered viscosity of the trastuzumab biosimilar at pH levels above 6.0, while glycine formulations were more effective at reducing viscosity below pH 6.0. Histidine-HCl, arginine-HCl, and lysine-HCl lowered the risk of HMW aggregation, whereas formulations containing Na-phosphate or NaCl showed higher HMW aggregation. Formulations with arginine-HCl, lysine-HCl, and NaCl demonstrated a rapid increase in fragmentation at pH levels below 5.0, while Na-aspartate formulations showed increased fragmentation at pH levels above 6.5. Conclusion: Hence, it is important to optimize the levels of each chosen excipient in the formulation study to balance their benefits against potential incompatibilities. This study serves as a foundation for identifying high-concentration antibody formulations using a high-throughput approach, where minimal materials are required, and optimized formulation design spaces can be quickly identified.

Microwave-Assisted Freeze-Drying: Impact of Microwave Radiation on the Quality of High-Concentration Antibody Formulations.

Microwave-assisted freeze-drying (MFD) offers significant time savings compared to conventional freeze-drying (CFD). While a few studies have investigated the stability of biopharmaceuticals with low protein concentrations after MFD and storage, the impact of MFD on high-concentration monoclonal antibody (mAb) formulations remains unclear. In this study, we systematically examined the effect of protein concentration in MFD and assessed protein stability following MFD, CFD, and subsequent storage using seven protein formulations with various stabilizers and concentrations. We demonstrated that microwaves directly interact with the active pharmaceutical ingredient (API), leading to decreased physical stability, specifically aggregation, in high-concentration antibody formulations. Furthermore, typically used sugar:protein ratios from CFD were insufficient for stabilizing mAbs when applying microwaves. We identified the intermediate drying phase as the most critical for particle formation, and cooling the samples provided some protection for the mAb. Our findings suggest that MFD technology may not be universally applicable to formulations well tested in CFD and could be particularly beneficial for formulations with low API concentrations requiring substantial amounts of glass-forming excipients, such as vaccines and RNA-based products.

Stable High-Concentration Monoclonal Antibody Formulations Enabled by an Amphiphilic Copolymer Excipient.

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

Ongoing Challenges to Develop High Concentration Monoclonal Antibody-based Formulations for Subcutaneous Administration: Quo Vadis?

Although many subcutaneously (s.c.) delivered, high-concentration antibody formulations (HCAF) have received regulatory approval and are widely used commercially, formulation scientists are still presented with many ongoing challenges during HCAF development with new mAb and mAb-based candidates. Depending on the specific physicochemical and biological properties of a particular mAb-based molecule, such challenges vary from pharmaceutical attributes e.g., stability, viscosity, manufacturability, to clinical performance e.g., bioavailability, immunogenicity, and finally to patient experience e.g., preference for s.c. vs. intravenous delivery and/or preferred interactions with health-care professionals. This commentary focuses on one key formulation obstacle encountered during HCAF development: how to maximize the dose of the drug? We examine methodologies for increasing the protein concentration, increasing the volume delivered, or combining both approaches together. We discuss commonly encountered hurdles, i.e., physical protein instability and solution volume limitations, and we provide recommendations to formulation scientists to facilitate their development of s.c. administered HCAF with new mAb-based product candidates.

Rapid Quantification of Protein Particles in High-Concentration Antibody Formulations

Current technologies for monitoring the subvisible particles that may be generated during fill-finish operations for protein formulations are cumbersome. Measurement times are generally too long for real-time analysis, and the high protein concentrations that are characteristic of many antibody products interfere with common optical techniques for particle analysis. To rapidly monitor protein particle levels in high-concentration protein solutions, we developed a fluorescence-based method that uses extrinsic fluorescent dyes such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid that are sensitive to the presence of aggregated protein. To test the method, antibody formulations containing various concentrations of protein particles were generated by application of various mechanical and freeze-thaw stresses. After addition of fluorescent dyes, fluorescence intensities were measured and compared to fluorescence intensities in particle-free formulations. The differences in fluorescence intensities were linearly proportional to protein particle levels, which for calibration purposes were measured offline by fluid imaging microscopy and protein assays. Protein particle levels could be measured without requiring sample dilution, even in high-concentration (e.g., 40 mg/mL) antibody formulations.

A Formulation Development Approach to Identify and Select Stable Ultra–High-Concentration Monoclonal Antibody Formulations With Reduced Viscosities

High protein concentration formulations are required for low-volume administration of therapeutic antibodies targeted for subcutaneous, self-administration by patients. Ultra-high concentrations (≥150 mg/mL) can lead to dramatically increased solution viscosities, which in turn can lead to stability, manufacturing, and delivery challenges. In this study, various categories and individual types of pharmaceutical excipients and other additives (56 in total) were screened for their viscosity reducing effects on 2 different mAbs. The physicochemical stability profile, as well as viscosity ranges, of several candidate antibody formulations, identified and designed based on the results of the excipient screening, were evaluated over a 6-month time period under accelerated and real-time storage conditions. In addition to reducing the solution viscosities to acceptable levels for parenteral administration (using currently available and acceptable delivery devices), the candidate formulations did not result in notable losses of physicochemical stability of the 2 antibodies on storage for 6 months at 25°C. The experiments described here demonstrate the feasibility of a formulation development and selection approach to identify candidate high-concentration antibody formulations with viscosities within pharmaceutically acceptable ranges that do not adversely affect their physicochemical storage stability.

Stress Tolerance of Antibody–Poly(Amino Acid) Complexes for Improving the Stability of High Concentration Antibody Formulations

The stabilization of antibodies in aqueous solution against physical stress remains a problematic issue for pharmaceutical applications. Recently, protein–polyelectrolyte complex (PPC) formation using poly(amino acids) was proposed to prepare antibody formulation in a salt-dissociable precipitated state without protein denaturation. Here, we investigated the stabilization effect of PPC of therapeutic antibodies with poly-l-glutamic acid on agitation and thermal stress as forms of mechanical and non-mechanical stress, respectively. The precipitated state of PPC prevented the inactivation and aggregation induced by agitation. Similar results were obtained using the suspension state of PPC, but the stabilizing effects were slightly inferior to those of the PPC precipitate. PPC precipitate and PPC suspension prevented heat-induced inactivation of the antibodies, but showed little effect on heat-induced aggregation. Thus, PPC is a new candidate as a simple storage method for antibodies in aqueous solution, as an alternative state for freeze-drying.

Feasibility of Antibody–Poly(Glutamic Acid) Complexes: Preparation of High-Concentration Antibody Formulations and Their Pharmaceutical Properties

Development of high-concentration antibody formulations for subcutaneous administration remains challenging. Recently, a precipitation-redissolution method was proposed to prepare suspensions or precipitates of salt-dissociable protein-poly(amino acid) complexes. To elucidate the utility of this method for protein therapy, we investigated the feasibility of a precipitation-redissolution method using poly(amino acid) for high-concentration antibody formulation. Omalizumab and adalimumab formulations of 150 mg/mL could be prepared using poly-l-glutamic acid (polyE) from low-concentration stock solutions. Enzyme-linked immunosorbent assay, circular dichroism, and size-exclusion chromatography revealed that the formation of antibody-polyE complex and precipitation-redissolution process did not significantly affect the immunoreactivity or secondary structure of the antibodies. The precipitation-redissolution method was less time-consuming and more effective than lyophilization-redissolution, evaporation-redissolution, and ultrafiltration from the viewpoint of final yield. Scalability was confirmed from 400 μL to 1.0 L. The general toxicity and pharmacokinetic profiles of the antibody-polyE complex formulations were similar to those of conventional antibody formulations. These results suggested that the precipitation-redissolution method using poly(amino acid) has great potential as a concentration method for antibody formulation and medicinal use.

Optimized UV Detection of High-Concentration Antibody Formulations using High-Throughput SE-HPLC

High-concentration antibody solutions (>100 mg/mL) present significant challenges for formulation and process development, including formulation attributes such as increased solution viscosity, and the propensity for self-association. An additional challenge comes from the adaptation of analytical methods designed for low-concentration formulations to the high-concentration regime. The oligomeric state is a good example: it is a quality attribute monitored during pharmaceutical development and is one that can be affected by dilution; a typical first step in the analysis of high-concentration solutions. The objective of this work was to develop a size-exclusion HPLC (SE-HPLC) method that would allow the injection of high-concentration antibody formulations without the need for dilution prior to injection and their analysis in a high-throughput manner that does not create a bottleneck for the execution of complex formulation development studies. It was found that changing the UV detection wavelength from 215 to 235 nm simplified sample preparation by allowing for an approximately fivefold increase in injection load while maintaining the signal within the linear range of detection. In addition, the chromatographic peak properties (i.e., peak symmetry, resolution, and sensitivity) were determined to be consistent when compared with analytical methods developed for formulations with lower antibody concentrations.

Surface Activity of a Monoclonal Antibody

The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4525–4533, 2009

Cross-Interaction Chromatography: A Rapid Method to Identify Highly Soluble Monoclonal Antibody Candidates

To develop a high-throughput cross-interaction chromatography screening method to rapidly identify antibody candidates with poor solubility using microgram quantities of purified material. A specific recombinant antibody or bulk polyclonal IgG purified from human serum was chemically coupled to an NHS-activated chromatography resin. The retention times of numerous monoclonal antibodies were determined on this resin using an HPLC and compared to the solubility of each antibody estimated by ultrafiltration. Retention times of the antibodies tested were found to be inversely related to solubility, with antibodies prone to precipitate at low concentrations in PBS being retained longer on the columns with broader peaks. The technique was successfully used to screen microgram quantities of a panel of therapeutic antibodies to identify candidates with low solubility in PBS. The cross-interaction chromatography methods described can be used to screen large panels of recombinant antibodies in order to discover those with low solubility. Addition of this tool to the array of tools available for characterization of affinity and activity of antibody therapeutic candidates will improve selection of candidates with biophysical properties favorable to development of high concentration antibody formulations.

Liquid high concentration IgG1 antibody formulations by precipitation

A manufacturing approach for liquid high concentration antibody formulations based on precipitation and subsequent re‐dissolution was investigated. IgG1 antibody solutions were concentrated from 20 to 100 mg/mL by intermediate precipitation, with a recovery exceeding 95%, retention of the native secondary structure and binding activity as well as adequate stability. Quantitative, reproducible precipitation was performed using 1.45 M ammonium sulphate (pH 5.5 and 8.0), 0.67 M sodium citrate (pH 8.0) and 9% (w/v) PEG 4000 (pH 5.5 and 8.0). Scalability was confirmed from 1 to 100 mL. The concentrations achievable in the re‐dissolution step were less affected by the re‐dissolution medium, but limited by the residual precipitant. Both, improved removal of remaining precipitant liquid and larger precipitation scales were successful in increasing the final protein concentration. SEC and turbidity analysis directly after re‐dissolution indicated that similar protein qualities were obtained, independent from the precipitant used. However, increased aggregate formation was observed after short term storage of the precipitated protein particles at either 2–8°C or ambient temperature. An accelerated mechanical and thermal stability program verified comparable stability of the re‐dissolved liquid 100 mg/mL formulations produced by intermediate precipitation to a control formulation obtained by standard ultrafiltration. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3043–3057, 2009

Viscoelastic characterization of high concentration antibody formulations using quartz crystal microbalance with dissipation monitoring

With increasing protein concentrations, therapeutic protein formulations are increasingly demonstrating significant deviations from ideal dilute solution behavior due to protein–protein interactions. These interactions lead to unique biophysical challenges in the administration of biopharmaceuticals including high apparent viscosity and viscoelasticity as well as challenges in maintaining the physical stability of proteins in solution. Here, we describe a straightforward analytical method to calculate the complex modulus and viscosity of high concentration protein solutions from measurements made using quartz crystal microbalance with dissipation monitoring (QCM-D). Further, this methodology was used to investigate the dependence of the storage and loss moduli (G′ and G″, respectively) of a humanized monoclonal antibody solution on solution pH. Unlike recent reports, the effect of protein deposition onto the surface of the quartz sensor crystal was measured and explicitly accounted for during analysis when determining the solution's complex modulus. It was found that the ratio G″/G′ was significantly greater than unity for all solutions investigated, but demonstrated a distinct maximum at pH 5.5 indicating that the solution exhibited the greatest liquid-like behavior at this pH. In addition, measurements were made at higher frequencies, which were found to be more sensitive to the changes in pH than those made at lower frequencies. It was also found that the viscoelastic ratio was relatively insensitive to the frequency of measurement at lower pH, but showed greater dependence on frequency as pH increased. The characterization of the rheological properties of high concentration antibody solutions provides insight into protein–protein interactions, and the methodology presented here demonstrates a straightforward way to determine the viscoelastic properties using ultrasonic rheology without the drawbacks of numerical fitting. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3108–3116, 2009