Monitoring of Visible Particles in Parenteral Products by Manual Visual Inspection-Reassessing Size Threshold and Other Particle Characteristics that Define Particle Visibility.

Visible particles are a critical quality attribute for parenteral products and must be monitored. A carefully designed, executed, and controlled drug product manufacturing process including a final 100 % visual inspection and appropriate end-product controls ensures that visible particles are consistently minimized and demonstrates that the injectable DP is practically free from visible particles. Visual inspection, albeit appearing as a simple analytical procedure, requires several technical and operational controls to ensure adequate performance. To gather new data on particle visibility and shed light on this decade-old challenge, a multi-company blinded visual inspection threshold study was conducted. A major goal of the study was visual assessment of several particle types of different sizes in small volume vials, as a challenging configuration for visual inspection, across 9 biopharmaceutical companies in order to determine the visibility limit. The study results provide key insights into limitations and challenges of visual inspection, namely, no universal visibility limit can be applied to all particle types as the detectability varies with particle type, number, and size. The study findings underscore the necessity of setting realistic expectations on size-based visibility limits in visual inspection, robust procedures for analyst training and qualification, and harmonization of guidelines globally.

Bacterial extracellular electron transfer components are spin selective.

Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway that spans from the inner membrane, across the periplasm, through the outer membrane, and to an external surface. Multiheme cytochromes are the primary machinery for moving electrons through this pathway. Recent studies show that the chiral-induced spin selectivity (CISS) effect is observable in some of these proteins extracted from the model metal-reducing bacteria, Shewanella oneidensis MR-1. It was hypothesized that the CISS effect facilitates efficient electron transport in these proteins by coupling electron velocity to spin, thus reducing the probability of backscattering. However, these studies focused exclusively on the cell surface electron conduits, and thus, CISS has not been investigated in upstream electron transfer components such as the membrane-associated MtrA, or periplasmic proteins such as small tetraheme cytochrome (STC). By using conductive probe atomic force microscopy measurements of protein monolayers adsorbed onto ferromagnetic substrates, we show that electron transport is spin selective in both MtrA and STC. Moreover, we have determined the spin polarization of MtrA to be ∼77% and STC to be ∼35%. This disparity in spin polarizations could indicate that spin selectivity is length dependent in heme proteins, given that MtrA is approximately two times longer than STC. Most significantly, our study indicates that spin-dependent interactions affect the entire extracellular electron transport pathway.

Efficient Spin-Selective Electron Transport at Low Voltages of Thia-Bridged Triarylamine Hetero[4]helicenes Chemisorbed Monolayer.

The Chirality Induced Spin Selectivity (CISS) effect describes the capability of chiral molecules to act as spin filters discriminating flowing electrons according to their spin state. Within molecular spintronics, efforts are focused on developing chiral-molecule-based technologies to control the injection and coherence of spin-polarized currents. Herein, for this purpose, we study spin selectivity properties of a monolayer of a thioalkyl derivative of a thia-bridged triarylamine hetero[4]helicene chemisorbed on a gold surface. A stacked device assembled by embedding a monolayer of these molecules between ferromagnetic and diamagnetic electrodes exhibits asymmetric magnetoresistance with inversion of the signal according to the handedness of molecules, in line with the presence of the CISS effect. In addition, magnetically conductive atomic force microscopy reveals efficient electron spin filtering even at unusually low potentials. Our results demonstrate that thia[4]heterohelicenes represent key candidates for the development of chiral spintronic devices.

A comprehensive strategy for the identification of biologics by liquid-chromatography-mass spectrometry for release testing in a regulated environment.

Identification (ID) testing is a regulatory requirement for biopharmaceutical manufacturing, requiring robust, GMP-qualified assays that can distinguish the therapeutic from any other in the facility. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical tool used to identify and characterize biologics. While routinely leveraged for characterization, LC-MS is relatively rare in Quality Control (QC) settings due to its perceived complexity and scarcity of MS-trained personnel. However, employing LC-MS for identification of drug products has many advantages versus conventional ID techniques, including but not limited to its high specificity, rapid turn-around time, and ease of method execution. In this work, we outline the development and implementation of a comprehensive LC-MS based ID strategy for biologics release testing. Two main workflows (WFs) were developed: i) WF1, a subunit-based assay measuring the molecular weight of the light chain (LC) and heavy chain (HC) of an antibody upon reduction, and ii) WF2, intact mass measurement of the biologic upon N-deglycosylation by PNGase F. The proposed strategy is shown to be applicable for over 40 diverse model biologics including monoclonal antibodies (mAbs), biobetters such as antibody prodrugs/afucosylated mAbs, fusion proteins, multi-specific antibodies, Fabs, and large peptides, all with excellent mass accuracy (error typically < 20 ppm) and precision. It requires a single-step sample preparation and a single click to run and process the data upon method setup. This strategy has been successfully implemented for release testing in GMP labs. Challenges and considerations for the establishment of QC-friendly methods are discussed. It is also shown that these methods can be applied to the ID of more analytically complex biotherapeutics, such as fixed-dose combination (FDC) and drug products co-formulated with trace-level additives.

Surface-Induced Protein Aggregation and Particle Formation in Biologics: Current Understanding of Mechanisms, Detection and Mitigation Strategies.

Protein stability against aggregation is a major quality concern for the production of safe and effective biopharmaceuticals. Amongst the different drivers of protein aggregation, increasing evidence indicates that interactions between proteins and interfaces represent a major risk factor for the formation of protein aggregates in aqueous solutions. Potentially harmful surfaces relevant to biologics manufacturing and storage include air-water and silicone oil-water interfaces as well as materials from different processing units, storage containers, and delivery devices. The impact of some of these surfaces, for instance originating from impurities, can be difficult to predict and control. Moreover, aggregate formation may additionally be complicated by the simultaneous presence of interfacial, hydrodynamic and mechanical stresses, whose contributions may be difficult to deconvolute. As a consequence, it remains difficult to identify the key chemical and physical determinants and define appropriate analytical methods to monitor and predict protein instability at these interfaces. In this review, we first discuss the main mechanisms of surface-induced protein aggregation. We then review the types of contact materials identified as potentially harmful or detected as potential triggers of proteinaceous particle formation in formulations and discuss proposed mitigation strategies. Finally, we present current methods to probe surface-induced instabilities, which represent a starting point towards assays that can be implemented in early-stage screening and formulation development of biologics.

Dual-detection approach for a charge variant analysis of monoclonal antibody combination products using imaged capillary isoelectric focusing.

The clinical benefits of treatments with a combination of two or more therapeutic monoclonal antibodies (mAbs) have emerged in recent years. Imaged capillary isoelectric focusing is a frequently used technology in the biopharmaceutical industry for charge variant analysis of protein therapeutics. However, with the wide concentration ranges of combination products, one component may fall within the linear detection range, whereas the other does not. Here, we report a novel methodology to explore charge variants of mAb mixtures using multiple detection techniques simultaneously. We use ultraviolet absorbance to monitor the charge variants of the high-concentration component and native fluorescence (FL) to monitor the variants of the low-concentration one. Charge variants of mixtures that span 40-fold in ratio differences can be accurately quantified with this approach. In contrast to the conventional methods, it is not necessary to prepare and analyze two samples at different concentrations and combine the results for combination product testing. Additionally, the use of FL detection enables the charge variant analysis of highly potent/low abundant mAbs in a mixture. This methodology is more quality-control friendly and efficient for the charge variant analysis of combination products with wide ratios.

Nucleation in Protein Aggregation in Biotherapeutic Development: A look into the Heart of the Event.

In spite of extensive research, protein aggregation still remains one of the most difficult phenomena to be understood in the field of biologics research and development. Protein aggregation is a complex process which results in the formation of a variety of supramolecular protein structures. Nucleation is the core step that initiates the cascade of molecular events leading to the formation of protein aggregates. Understanding and characterizing nucleation is therefore crucial to avoid undesired protein aggregation. Here we review the state of the art on protein aggregation in biotherapeutics, primarily focusing on the nucleation events, stimulating discussions about key open questions, and clarifying the peculiarities of aggregation process relative to other protein phase separation processes, such as crystallization. We summarize recent progress in the identification of the sources of protein aggregation and in the development of analytical tools to characterize this process. Moreover, we discuss significant gaps in the analysis and understanding of nucleation in non-native aggregation of biologics.

Stress Factors in Primary Packaging, Transportation and Handling of Protein Drug Products and Their Impact on Product Quality.

Protein-based biologic drugs encounter a variety of stress factors during drug substance (DS) and drug product (DP) manufacturing, and the subsequent steps that result in clinical administration by the end user. This article is the third in a series of commentaries on these stress factors and their effects on biotherapeutics. It focuses on assessing the potential negative impact from primary packaging, transportation, and handling on the quality of the DP. The risk factors include ingress of hazardous materials such as oxidizing residuals from the sterilization process, delamination- or rubber stopper-derived particles, silicone oil droplets, and leachables into the formulation, as well as surface interactions between the protein and packaging materials, all of which may cause protein degradation. The type of primary packaging container used (such as vials and prefilled syringes) may substantially influence the impact of transportation and handling stresses on DP Critical Quality Attributes (CQAs). Mitigations via process development and robustness studies as well as control strategies for DP CQAs are discussed, along with current industry best practices for scale-down and in-use stability studies. We conclude that more research is needed on postproduction transportation and handling practices and their implications for protein DP quality.

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality.

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

Emerging Challenges and Innovations in Surfactant-mediated Stabilization of Biologic Formulations.

Biologics may be subjected to various destabilizing conditions during manufacturing, transportation, storage, and use. Therefore, biologics must be appropriately formulated to meet their desired quality target product profiles. In the formulations of protein-based biologics, one critical component is surfactant. Polysorbate 80 and Polysorbate 20 remain the most commonly used surfactants. Surfactants can stabilize proteins through different mechanisms and help the proteins withstand destabilization stresses. However, the challenges associated with surfactants, for instance, impurities, degradation, and potential triggering of adverse immune responses, have been encountered. Therefore, there are continued efforts to develop novel surfactants to overcome these challenges associated with traditional surfactants. Meanwhile, surfactants have also found their use in formulations of newer and novel modalities, namely, antibody-drug conjugates, bispecific antibodies, and adeno-associated viruses (AAV). This review provides an updated in-depth discussion of surfactants in the above-mentioned areas, namely mechanism of action of surfactants, a critical review of challenges with surfactants and current mitigation approaches, and emerging technologies to develop novel surfactants. In addition, gaps, current mitigations, and future directions have been presented to trigger further discussion and research to facilitate the use and development of novel surfactants.

Mimicking Low pH Virus Inactivation Used in Antibody Manufacturing Processes: Effect of Processing Conditions and Biophysical Properties on Antibody Aggregation and Particle Formation.

Low pH virus inactivation (VI) step is routinely used in antibody production manufacturing. In this work, a mimic of the VI step was developed to focus on evaluating adverse effects on product quality. A commercially available lab-scale glass reactor system was utilized to assess impacts of process and solution conditions on process-induced monoclonal antibody particle formation. Flow imaging was found to be more sensitive than light obscuration in detecting microparticles. NaOH as a base titrant increased protein microparticles more than Tris. Both stirring and NaCl accelerated particle formation, indicating that interfacial stress and protein colloidal stability were important factors. Polysorbate 80 was effective at suppressing particle formation induced by stirring. In contrast, trehalose led to higher microparticle levels suggesting a conformational stabilizer may have other adverse effects during titration with stirring. Additionally, conformational and colloidal stability of antibodies were characterized to investigate the potential roles of antibody physicochemical properties in microparticle formation during VI. The stability data were supportive in rationalizing particle formation behaviors, but they were not predictive of particle formation during the mimicked viral inactivation steps. Overall, the results demonstrate the value of testing various solution and processing conditions in a scaled-down system prior to larger-scale VI bioprocesses.

A Detailed Protocol for Generation of Therapeutic Antibodies with Galactosylated Glycovariants at Laboratory Scale Using In-Vitro Glycoengineering Technology.

N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG.

Bridging size and charge variants of a therapeutic monoclonal antibody by two-dimensional liquid chromatography.

Monoclonal antibodies are heterogeneous in nature and may contain numerous variants with differences in size, charge, and hydrophobicity, which may impact clinical efficacy, immunogenicity, and safety. Characterization of antibody variants is necessary to build structure-function correlation and establish a proper control strategy. Isolation and enrichment of variants by conventional chromatographic peak fractionation is labor-intensive and time-consuming. The instability of fractions during isolation and subsequent characterization may also be a concern. Hence, it is desirable to analyze antibody variants in an online and real-time manner. Here we demonstrate a 2D-LC methodology - multiple heart-cutting IEC-SEC- as an investigational tool to facilitate a charge variant characterization study. Both IEC modes - anion exchange (AEX) and cation exchange (CEX) chromatography are discussed. Using this approach, direct bridging of size and charge variants of an antibody molecule was achieved without offline peak fractionation. It was observed that antibody aggregates elute late on both the AEX and CEX columns, presumably due to secondary hydrophobic interactions. Additionally, we overcame the solvent mismatch issue and developed a 2D SEC-IEC method to confirm the bridging results. This is the first reported SEC-IEC 2D-LC application for the characterization of antibody size and charge variants.

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy.

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations.

Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit.

Isomerization of surface-exposed aspartic acid (Asp) in the complementarity-determining regions of therapeutic proteins could potentially impact their target binding affinity because of the sensitive location, and often requires complex analytical tactics to understand its effect on structure-function and stability. Inaccurate quantitation of Asp-isomerized variants, especially the succinimide intermediate, presents major challenge in understanding Asp degradation kinetics, its stability, and consequently establishing a robust control strategy. As a practical solution to this problem, a comprehensive analytical tool kit has been developed, which provides a solution to fully characterize and accurately quantify the Asp-related product variants. The toolkit offers a combination of 2 steps, an ion-exchange chromatography method to separate and enrich the isomerized variants in the folded structure for structure-function evaluation and a novel focused peptide mapping method to quantify the individual complementarity-determining region isomerization components including the unmodified Asp, succinimide, and isoaspartate. This novel procedure allowed an accurate quantification of each Asp-related variant and a comprehensive assessment of the functional impact of Asp isomerization, which ultimately helped to establish an appropriate control strategy for this critical quality attribute.

Unique Impacts of Methionine Oxidation, Tryptophan Oxidation, and Asparagine Deamidation on Antibody Stability and Aggregation.

Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility, and aggregation. Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and nonreducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. These findings highlight that even related types of chemical modifications can lead to dissimilar antibody aggregation mechanisms, and evaluating these findings for additional antibodies will be important for improving the systematic generation of antibodies with high chemical and physical stability.

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 µm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique.

Fragmentation in protein-based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size-exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE-SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein-based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab-related low molecular weight fragments, a non-reducible thio-ether linked 75 kDa HL fragment was also identified.