• Adsorption
• Biomanufacture
• Biopharmaceutical stability
• Interfaces
• Mitigation
• Particles
• Primary packaging

• IgG
• antibody
• antibody stability
• antibody tests
• clinical specimens
• veterinary diagnostic medicine

• Immunoglobulin G/blood
• Animals
• Specimen Handling

• Antibody-drug conjugate
• Column performance optimization
• Monoclonal antibody
• Protein-protein interaction
• Reversible self-association
• Size exclusion chromatography

• Immunoconjugates/chemistry
• Chromatography, Gel/methods
• Hydrophobic and Hydrophilic Interactions
• Antibodies, Monoclonal/chemistry
• Chromatography, High Pressure Liquid/methods
• Chromatography, Reverse-Phase/methods
• Arginine/chemistry
• Acetonitriles/chemistry

• LHDMZ24H300003 Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China
• 2023-1-120 Jinhua Science and Technology Plan Projects
• 2024TZSX105 Municipal-University Cooperation Projects of Taizhou Institute of Zhejiang University
• XY2023016 Scientific Research Fund of Zhejiang University
• 24gya05 Taizhou Science and Technology Plan Projects

• Image analysis
• Machine learning
• Microparticles
• Neural networks
• Protein aggregation
• Protein formulation

• Antibodies, Monoclonal/chemistry
• Machine Learning
• Drug Packaging/methods
• Protein Aggregates
• Particle Size
• Drug Compounding/methods
• Drug Storage
• Neural Networks, Computer
• Drug Stability
• Freezing
• Chemistry, Pharmaceutical/methods
• Protein Stability

• Dynamic Nuclear Polarization
• crystalline suspension
• frozen solution
• low-temperature NMR spectroscopy
• monoclonal antibody
• pembrolizumab
• phase transition
• stability

• antibody–drug conjugate
• mobile phase
• monoclonal antibody
• secondary interaction
• size exclusion chromatography
• sodium iodide

• Antibodies, Monoclonal/chemistry
• Chromatography, Gel/methods
• Immunoconjugates/chemistry
• Sodium Iodide/chemistry
• Osmolar Concentration
• Hydrophobic and Hydrophilic Interactions
• Hydrogen-Ion Concentration
• Sodium Chloride/chemistry
• Static Electricity
• Arginine/chemistry

• 2024TZSX105 Municipal-University Cooperation Projects of Taizhou Institute of Zhejiang University
• LHDMZ24H300003 Huadong Medicine Joint Funds of the Zhejiang Provincial Natural Science Foundation of China
• 2023-1-120 Jinhua Science and Technology Plan Projects

• cryo-concentration
• drug substance bottles
• frozen storage
• large-scale freezing
• process characterization

• Freezing
• Ice
• Drug Packaging/methods
• Osmolar Concentration
• Polysorbates/chemistry
• Histidine/chemistry
• Biological Products/chemistry

• biopharmaceutical
• gas chromatography
• genotoxic impurity
• liquid chromatography
• mass spectrometry
• pharmaceutical analysis

• Drug Discovery
• Drug Development
• Biological Products/chemistry
• Mass Spectrometry/methods
• Pharmaceutical Preparations

• Chaperon
• Disaggregase
• Extrinsic factors
• Intrinsic factors
• Protein aggregation

• Amyloid/metabolism
• Humans
• Animals
• Protein Aggregates

• In-line monitoring
• Machine learning
• Process analytical technology
• Process robustness
• Therapeutic antibody

• Spectrum Analysis, Raman
• Chromatography
• Calibration
• Pharmaceutical Preparations
• Technology, Pharmaceutical/methods

• Freeze–thaw treatment
• Precipitation
• Purification
• Recombinant protein
• RuBisCO

• Plant Proteins/genetics
• Plant Proteins/metabolism
• Ribulose-Bisphosphate Carboxylase/metabolism
• Proteomics/methods
• Freezing
• Photosynthesis/physiology
• Plants/metabolism
• Recombinant Proteins/metabolism
• Plant Extracts
• Plant Leaves/metabolism
• Carbon Dioxide/metabolism

• Biologics
• Drug product
• Formulation
• Formulation robustness
• Monoclonal antibodies
• Process characterization
• Process development
• Process validation
• QbD
• Roadmap
• Target product profile

• Humans
• Biological Products
• Drug Development
• Drug Industry

• Accelerated stress
• NMR
• biologics
• drug product stability
• high-concentration drug product
• mABs
• protein aggregation
• wNMR

• Drug Industry/methods
• Viscosity
• Water/chemistry
• Magnetic Resonance Spectroscopy

• Aggregation
• Bioprocessing
• Bulk freezing
• Chemical instability
• Fermentation
• Manufacturing
• Protein stability
• Protein therapeutics
• Purification

• Brij 35
• Interfaces
• Monoclonal antibody(s)
• Poloxamer 188
• Polysorbates
• Protein aggregation
• Protein formulation(s)
• Stability
• Surfactant

• Biopharmaceuticals
• Enzymes
• Gene therapy
• Insulin
• Monoclonal antibodies
• Nanotechnology
• Protein aggregation
• Recombinant proteins
• Stability
• Vaccines

• Lysine
• Antibodies, Monoclonal
• Imines
• Mass Spectrometry
• Serine

• Regeneron Pharmaceuticals

• Antibody
• Biologic
• Commercial
• Formulation
• Isoelectric point
• pH

• Humans
• Antibodies, Monoclonal/chemistry
• Isoelectric Point
• Arginine/chemistry
• Excipients/chemistry
• Sucrose/chemistry

• GLA
• NAFLD
• gut microbiota
• inflammatory cytokines
• intestinal permeability
• probiotics
• tight junction

• Freeze-concentration
• Freeze-thaw
• Ice surface area
• Protein stability

• Freezing
• L-Lactate Dehydrogenase/metabolism
• Ice
• Histidine
• Proteins

• Antibodies
• Formulation
• Particles
• Protein aggregation
• Surface

• Protein Aggregates
• Chemistry, Pharmaceutical/methods
• Biological Products
• Membrane Proteins
• Water

• Analysis
• Database
• Particle size
• Protein aggregation
• Stability

• Aggregation
• Formulation
• Freeze and thaw damage
• Frozen mab solutions
• Raman spectroscopy
• Secondary structure
• Tertiary structure
• Tyrosine ratio

• Antibody
• biologics
• colloidal
• conformational
• developability
• formulation
• manufacturability
• specificity
• stability

• Biological Products/therapeutic use
• Antibodies
• Risk Assessment
• Workflow

Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at −80 °C, −20 °C, and − 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure T_g′ of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine T_g′ in formulations without glass forming sugars. Storage below T_g′ did not lead to mAb aggregation. Above T_g′, at −20 °C and − 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below T_g′ completely prevents mAb aggregation and particle formation.

We examined the impact of monoclonal antibody (mAb) and buffer concentration, mimicking the cryoconcentration found upon freezing in a 2 L bottle, on mAb stability during frozen storage. Upon cryoconcentration, larger protein molecules and small excipient molecules freeze-concentrate differently, resulting in different protein to stabiliser ratios within a container. Understanding the impact of these shifted ratios on protein stability is essential. For two mAbs a set of samples with constant mAb (5 mg/mL) or buffer concentration (medium histidine/adipic acid) was prepared and stored for 6 months at −10 °C. Stability was evaluated via size-exclusion chromatography, flow imaging microscopy, UV/Vis spectroscopy at 350 nm, and protein A chromatography. Dynamic light scattering was used to determine k_D values. Soluble aggregate levels were unaffected by mAb concentration, but increased with histidine concentration. No trend in optical density could be identified. In contrast, increasing mAb or buffer concentration facilitated the formation of subvisible particles. A trend towards attractive protein-protein interactions was seen with higher ionic strength. MAb oxidation levels were negatively affected by increasing histidine concentration, but became less with higher mAb concentration. Small changes in mAb and buffer composition had a significant impact on stability during six-month frozen storage. Thus, preventing cryoconcentration effects in larger freezing containers may improve long-term stability.

• antibody
• antibody-drug conjugate
• bispecific
• developability
• discovery

Regulatory authorities place stringent guidelines on the removal of contaminants during the manufacture of biopharmaceutical products. Monoclonal antibodies, Fc-fusion proteins, and other mammalian cell-derived biotherapeutics are heterogeneous molecules that are validated based on the production process and not on molecular homogeneity. Validation of clearance of potential contamination by viruses is a major challenge during the downstream purification of these therapeutics. Virus filtration is a single-use, size-based separation process in which the contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filtration is routinely used as part of the overall virus clearance strategy. Compromised performance of virus filters due to membrane fouling, low throughput and reduced viral clearance, is of considerable industrial significance and is frequently a major challenge. This review shows how components generated during cell culture, contaminants, and product variants can affect virus filtration of mammalian cell-derived biologics. Cell culture-derived foulants include host cell proteins, proteases, and endotoxins. We also provide mitigation measures for each potential foulant.

• Aggregation
• Freezing
• Monoclonal antibody
• Scale-down device
• Thawing

• Concentration gradient
• Freeze–thaw
• Large-scale thawing
• Monoclonal antibody
• Scale-down device

High physical stability is required for the development of monoclonal antibodies (mAbs) into successful therapeutic products. Developability assays are used to predict physical stability issues such as high viscosity and poor conformational stability, but protein aggregation remains a challenging property to predict. Among different types of stresses, air–water and solid–liquid interfaces are well known to potentially trigger protein instability and induce aggregation. Yet, in contrast to the increasing number of developability assays to evaluate bulk properties, there is still a lack of experimental methods to evaluate antibody stability against interfaces. Here, we investigate the potential of a hydrophobic nanoparticle surface-mediated stress assay to assess the stability of mAbs during the early stages of development. We evaluate this surface-mediated accelerated stability assay on a rationally designed library of 14 variants of a humanized IgG4, featuring a broad span of solubility values and other developability properties. The assay could identify variants characterized by high instability against agitation in the presence of air–water interfaces. Remarkably, for the set of investigated molecules, we observe strong correlations between the extent of aggregation induced by the surface-mediated stress assay and other developability properties of the molecules, such as aggregation upon storage at 45°C, self-association (evaluated by affinity-capture self-interaction nanoparticle spectroscopy) and nonspecific interactions (estimated by cross-interaction chromatography, stand-up monolayer chromatography (SMAC), SMAC*). This highly controlled surface-mediated stress assay has the potential to complement and increase the ability of the current set of screening techniques to assess protein aggregation and developability potential of mAbs during the early stages of drug development.

Abbreviations:AC-SINS: Affinity-Capture Self-Interaction Nanoparticle Spectroscopy; AMS: Ammonium sulfate precipitation; ANS: 1-anilinonaphtalene-8-sulfonate; CIC: Cross-interaction chromatography; DLS: Dynamic light scattering; HIC: Hydrophobic interaction chromatography; HNSSA: Hydrophobic nanoparticles surface-stress assay; mAb: Monoclonal antibody; NP: Nanoparticle; SEC: Size exclusion chromatography; SMAC: Stand-up monolayer chromatography; WT: Wild type

• aggregation
• antibodies
• developability
• formulation
• interfaces
• stability
• surfaces

• Protein stability
• aggregation
• excipients
• mechanism
• modelling

Physical instabilities of proteins in the form of protein aggregation continue to be a major challenge in the development of protein drug candidates. Aggregation can occur during different stages of product lifecycle such as freeze–thaw, manufacturing, shipping, and storage, and can potentially delay commercialization of candidates. A lack of clear understanding of the underlying mechanism(s) behind protein aggregation and the potential immunogenic reactions renders the presence of aggregates in biotherapeutic products undesirable. Understanding and minimizing aggregation can potentially reduce immunogenic responses and make protein therapeutics safer. Therefore, it is imperative to identify, understand, and control aggregation during early formulation development and develop reliable and orthogonal analytical methodologies to detect and monitor levels of aggregation. Freezing and thawing are typical steps involved in the manufacturing of drug product and could result in complex physical and chemical changes, which in turn could potentially cause protein aggregation. This study provides a systematic approach in understanding and selecting the ideal freeze–thaw conditions for manufacturing of protein-based therapeutics. It identifies the importance of balancing different excipients with an overall goal of sufficiently reducing or eliminating aggregation and developing a stable and scalable formulation. The results demonstrated that the freeze–thaw damage of mAb-1 in aqueous solutions was significantly reduced by identification of optimal freeze–thaw conditions using first a small-scale model with subsequent at-scale verifications. The work provides a framework for successful transfer of drug product manufacturing process from small-scale to the manufacturing scale production environment especially for molecules that are susceptible to freeze–thaw induced degradations.

• Biotechnology and Biological Sciences Research Council
• Bristol-Myers Squibb

Therapeutic proteins as biopharmaceuticals have emerged as a very important class of drugs for the treatment of many diseases. However, they are less stable compared to conventional pharmaceuticals. Their long-term stability in solid forms, which is critical for product performance, depends heavily on the retention of the native protein structure during the lyophilization (freeze-drying) process and, thereafter, in the solid state. Indeed, the biological function of proteins is directly related to the tertiary and secondary structure. Besides physical stability and biological activity, conformational stability (three-dimensional structure) is another important aspect when dealing with protein pharmaceuticals. Moreover, denaturation as loss of higher order structure is often a precursor to aggregation or chemical instability. Careful study of the physical and chemical properties of proteins in the dried state is therefore critical during biopharmaceutical drug development to deliver a final drug product with built-in quality that is safe, high-quality, efficient, and affordable for patients. This review provides an overview of common analytical techniques suitable for characterizing pharmaceutical protein powders, providing structural, and conformational information, as well as insights into dynamics. Such information can be very useful in formulation development, where selecting the best formulation for the drug can be quite a challenge.

• analytical tools
• antibody
• excipients
• formulation development
• lyophilization
• protein characterization
• protein structure
• safe drug
• solid pharmaceuticals
• stable drug product

• Drug stability
• PCSK9 monoclonal antibody
• Temperature

• regulatory guideline
• Monoclonal antibodies
• aggregation
• stability
• therapeutics

Detection and characterization of particles in the visible and subvisible size range is critical in many fields of industrial research. Commercial particle analysis systems have proliferated over the last decade. Despite that growth, most systems continue to be based on well-established principles, and only a handful of new approaches have emerged. Identifying the right particle-analysis approach remains a challenge in research and development. The choice depends on each individual application, the sample, and the information the operator needs to obtain. In biopharmaceutical applications, particle analysis decisions must take product safety, product quality, and regulatory requirements into account. Biopharmaceutical process samples and formulations are dynamic, polydisperse, and very susceptible to chemical and physical degradation: improperly handled product can degrade, becoming inactive or in specific cases immunogenic. This article reviews current methods for detecting, analyzing, and characterizing particles in the biopharmaceutical context. The first part of our article represents an overview about current particle detection and characterization principles, which are in part the base of the emerging techniques. It is very important to understand the measuring principle, in order to be adequately able to judge the outcome of the used assay. Typical principles used in all application fields, including particle–light interactions, the Coulter principle, suspended microchannel resonators, sedimentation processes, and further separation principles, are summarized to illustrate their potentials and limitations considering the investigated samples. In the second part, we describe potential technical approaches for biopharmaceutical particle analysis as some promising techniques, such as nanoparticle tracking analysis (NTA), micro flow imaging (MFI), tunable resistive pulse sensing (TRPS), flow cytometry, and the space- and time-resolved extinction profile (STEP^®) technology.

• biopharmaceuticals
• emerging particle techniques
• particle characterization
• particle detection
• particle quantification
• protein aggregates

• ATR
• Aggregation
• Biopharmaceuticals
• Bioprocessing
• FTIR spectroscopy
• Proteins

• Biosimilar Pharmaceuticals
• Diagnostic Imaging
• Protein Structure, Secondary
• Proteins
• Spectroscopy, Fourier Transform Infrared

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens.

To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates.

This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

• Analytical biochemistry
• Circular dichroism
• Light scattering (dynamic)
• Lyophilization
• Mucosal immunization
• Mucosal vaccination
• Oral drug delivery
• Physicochemical properties
• Preformulation
• Protease
• Protein aggregation
• Protein formulation
• Stability
• Vaccine adjuvants

• antineoplastic activity
• in-use stability
• microbiological stability
• monoclonal antibodies
• pharmaceutical preparation
• physicochemical stablity

• image analysis
• machine learning
• protein aggregation
• protein formulation

• Algorithms
• Antibodies/analysis
• Antibodies/chemistry
• Antibodies/metabolism
• Image Processing, Computer-Assisted/methods
• Immunoglobulins, Intravenous/analysis
• Immunoglobulins, Intravenous/chemistry
• Immunoglobulins, Intravenous/metabolism
• Machine Learning
• Microscopy/methods
• Protein Aggregates
• Protein Stability

• R41 GM130513 NIGMS NIH HHS