Scaling Down Large-Scale Thawing of Monoclonal Antibody Solutions: 3D Temperature Profiles, Changes in Concentration, and Density Gradients.

PURPOSE: Scale-down devices (SDD) are designed to simulate large-scale thawing of protein drug substance, but require only a fraction of the material. To evaluate the performance of a new SDD that aims to predict thawing in large-scale 2 L bottles, we characterised 3D temperature profiles and changes in concentration and density in comparison to 125 mL and 2 L bottles. Differences in diffusion between a monoclonal antibody (mAb) and histidine buffer after thawing were examined. METHODS: Temperature profiles at six distinct positions were recorded with type T thermocouples. Size-exclusion chromatography allowed quantification of mAb and histidine. Polysorbate 80 was quantified using a fluorescent dye assay. In addition, the solution's density at different locations in bottles and the SDD was identified. RESULTS: The temperature profiles in the SDD and the large-scale 2 L bottle during thawing were similar. Significant concentration gradients were detected in the 2 L bottle leading to marked density gradients. The SDD slightly overestimated the dilution in the top region and the maximum concentrations at the bottom. Fast diffusion resulted in rapid equilibration of histidine. CONCLUSION: The innovative SDD allows a realistic characterisation and helps to understand thawing processes of mAb solutions in large-scale 2 L bottles. Only a fraction of material is needed to gain insights into the thawing behaviour that is associated with several possible detrimental limitations.

Cryoconcentration and 3D Temperature Profiles During Freezing of mAb Solutions in Large-Scale PET Bottles and a Novel Scale-Down Device.

PURPOSE: Small-scale models that simulate large-scale freezing of bulk drug substance of biopharmaceuticals are highly needed to define freezing and formulation parameters based on process understanding. We evaluated a novel scale-down device (SDD), which is based on a specially designed insulation cover, with respect to changes in concentration after freezing, referred to as cryoconcentration, and 3D temperature profiles. Furthermore, the effect of the initial monoclonal antibody (mAb) concentration on cryoconcentration was addressed. METHODS: 2 L and 125 mL bottles were utilized. Temperatures were mapped using type T thermocouples. Frozen blocks were cut and mAb and histidine concentrations were analysed by HPLC. In addition, concentration- and temperature-dependent viscosities were measured. RESULTS: 3D freezing profiles in the SDD were comparable to large-scale bottles. The SDD accurately predicted cryoconcentration of both mAb and histidine of large-scale freezing. Concentric changes in concentration were evident as well as an unforeseen diluted core at the last point to freeze. At low initial mAb concentration cryoconcentration was substantial, while high initial mAb concentration suppressed cryoconcentration almost completely. CONCLUSION: The novel SDD gives detailed insights into large-scale freezing of mAb solutions using only a fraction of the simulated volume. It is a promising material- and cost-saving tool to understand large-scale freezing processes.

The silicone depletion in combination products induced by biologics.

Silicone oil (SO) migration into the drug product of combination products for biopharmaceuticals during storage is a common challenge. As the inner barrel surface is depleted of SO the extrusion forces can increase compromising the container functionality. In this context we investigated the impact of different formulations on the increase in gliding forces in a spray-on siliconized pre-filled syringe upon storage at 2-8 °C, 25 °C and 40 °C for up to 6 months. We tested the formulation factors such as surfactant type, pH, and ionic strength in the presence of one monoclonal antibody (mAb) as well as compared three mAbs in one formulation. After 1 month at 40 °C, the extrusion forces were significantly increased due to SO detachment dependent on the fill medium. The storage at 40 °C enhanced the SO migration process but it could also be observed at lower storage temperatures. Regarding the formulation factors the tendency for SO migration was predominantly dependent on the presence and type of surfactant. Interestingly, when varying the mAb molecules, one of the proteins showed a rather stabilizing effect on the SO layer resulting into higher container stability. In contrast to the formulation factors, those different stability outcomes could not be explained by interfacial tension (IFT) measurements at the SO interface. Further characterization of the mAb molecules regarding interfacial rheology and conformational stability were not adequately able to explain the observed difference. Solely a hydrophobicity ranking of the molecules correlated to the stability outcome. Further investigations are needed to clarify the role of the protein in the SO detachment process and to understand the cause for the stabilization. However, the study clearly demonstrated that the protein itself plays a critical role in the SO detachment process and underlined the importance to include verum for container stability.

Modular and tunable alternative surfactants for biopharmaceuticals provide insights into Surfactant's Structure-Function relationship.

Surface-induced aggregation of protein therapeutics is opposed by employing surfactants, which are ubiquitously used in drug product development, with polysorbates being the gold standard. Since poloxamer 188 is currently the only generally accepted polysorbate alternative, but cannot be ubiquitously applied, there is a strong need to develop surfactant alternatives for protein biologics that would complement and possibly overcome known drawbacks of existing surfactants. Yet, a severe lack of structure-function relationship knowledge complicates the development of new surfactants. Herein, we perform a systematic analysis of the structure-function relationship of three classes of novel alternative surfactants. Firstly, the mode of action is thoroughly characterized through tensiometry, calorimetry and MD simulations. Secondly, the safety profiles are evaluated through cell-based in vitro assays. Ultimately, we could conclude that the alternative surfactants investigated possess a mode of action and safety profile comparable to polysorbates. Moreover, the biophysical patterns elucidated here can be exploited to precisely tune the features of future surfactant designs.

Risk-based approach to setting sterile filtration microbial bioburden limits - Focus on biotech-derived products.

Holistic concepts should be applied that reduce risks prior to final bioburden testing and sterile filtration, based on enhanced process and product attribute understanding, which could be key to successful bioburden risk management. Key findings of this paper include.

Visualisation and quantification of subcutaneous injections of different volumes, viscosities and injection rates: An ex-vivo micro-CT study.

The effects of subcutaneous (SC) injection parameters such as drug formulation volume, viscosity and injection rate on therapeutic performance and tolerability have not been established for any drug product. In this study four groups of SC injections were performed on fresh ex vivo minipig abdominal tissue samples, varying volume (0.5-1 mL), viscosity (1-11 cP) and rate (0.02-0.1 mL/s). Micro-CT provided high resolution (50 micron) imaging of the SC tissues before and after injection, enabling a detailed 3D visualisation and analysis of how both injection parameters and tissue microstructure influence spatial distribution of injectables. We found that volume was the only significant factor for spatial distribution of injectate within our design space, and there were no significant factors for tissue backpressure. Variability within test groups was typically greater than differences between group means. Accordingly, whilst the higher viscosity formulations consistently exhibited reduced spatial distribution, the sample size was not large enough to establish confidence in this result. Comparing our findings to clinical evidence, we conclude that injection site and depth are more likely to influence PK and bioavailability than volume, viscosity and rate within our experimental space.

Evaluation of a novel silicone oil free primary packaging system with PTFE-based barrier stopper for biologics.

In order to overcome silicone oil related problems for biopharmaceuticals, novel container systems are of interest with a focus on the reduction, fixation or complete avoidance of silicone oil in the primary container. Ultimately, silicone oil free (SOF) container systems made from cyclic olefin (co-)polymer or glass combined with the respective silicone-oil free plungers were developed. In the following study we evaluated the potential of a SOF container system based on a glass barrel in combination with a fluoropolymer coated syringe plunger. In a long-term stability study, the system was compared to other alternative container systems in terms of functionality and particle formation when filled with placebo buffers. The system proved to be a valuable alternative to marketed siliconized container systems with acceptable and consistent break-loose gliding forces and it was clearly superior in terms of particle formation over storage time. Additionally, we evaluated the importance of the glass barrel surface for functionality. The interaction of the fill medium with the glass surface significantly impacted friction forces. Consequently, storage conditions and production processes like washing and sterilization, which can easily alter the surface properties, should be carefully evaluated, and controlled. The novel combination of non-lubricated glass barrel and fluoropolymer coated plunger provides a highly valuable SOF packaging alternative for biopharmaceuticals.

Impact of autoclavation on baked-on siliconized containers for biologics.

Many pharmaceutical manufacturing units utilize pre-sterilized ready-to fill primary containers for parenterals. The containers may have been sterilized by the supplier via autoclavation. This process can change the physicochemical properties of the material and the subsequent product stability. We studied the impact of autoclavation on baked on siliconized glass containers for biopharmaceuticals. We characterized the container layers of different thickness before and after autoclavation for 15 min at 121 °C and 130 °C. Furthermore, we analyzed the adsorption of a mAb to the silicone layer and subjected filled containers to 12 weeks storage at 40 °C monitoring functionality and subvisible particle formation of the product. Autoclavation turned the initially homogenous silicone coating into an incoherent surface with uneven microstructure, changed surface roughness and energy, and increased protein adsorption. The effect was more pronounced at higher sterilization temperatures. We did not observe an effect of autoclavation on stability. Our results did not indicate any concerns for autoclavation at 121 °C for safety and stability of drug/device combination products using baked-on siliconized glass containers.

Raman Marker Bands for Secondary Structure Changes of Frozen Therapeutic Monoclonal Antibody Formulations During Thawing.

In this work we use Raman spectroscopy for protein characterization in the frozen state. We investigate the behavior of frozen therapeutic monoclonal antibody IgG1 formulation upon thawing by Raman spectroscopy. Secondary and tertiary structure of the protein in three different mab formulations in the frozen state are followed through observation of marker bands for α-helix, ß-sheet and random coil. We identify the tyrosine intensity ratio I856/I830 as a marker for mab aggregation. Upon fast cooling (40 °C/min) to -80 °C we observe a significant increase of random coil and α -helical structures, while this is not the case for slower cooling (20 °C/min) to -80 °C. Most changes in the protein's secondary structure are observed in the course of thawing in the range up to -20 °C, when passing through the glass transitions and cold-crystallization of the two types of freeze-concentrated solutions formed through macro- and microcryoconcentration. An increase of protein concentration and the addition of mannitol suppress secondary structural changes but do no impact on aggregation.

Not the Usual Suspects: Alternative Surfactants for Biopharmaceuticals.

Therapeutically relevant proteins naturally adsorb to interfaces, causing aggregation which in turn potentially leads to numerous adverse consequences such as loss of activity or unwanted immunogenic reactions. Surfactants are ubiquitously used in biotherapeutics drug development to oppose interfacial stress, yet, the choice of the surfactant is extremely limited: to date, only polysorbates (PS20/80) and poloxamer 188 are used in commercial products. However, both surfactant families suffer from severe degradation and impurities of the raw material, which frequently increases the risk of particle generation, chemical protein degradation, and potential adverse immune reactions. Herein, we assessed a total of 40 suitable alternative surfactant candidates and subsequently performed a selection through a three-gate screening process employing four protein modalities encompassing six different formulations. The screening is based on short-term agitation-induced aggregation studies coupled to particle analysis and surface tension characterization, followed by long-term quiescence stability studies connected to protein purity measurements and particle analysis. The study concludes by assessing the surfactant's chemical and enzymatic degradation propensity. The candidates emerging from the screening are de novo α-tocopherol-derivatives named VEDG-2.2 and VEDS, produced ad hoc for this study. They display protein stabilization potential comparable or better than polysorbates together with an increased resistance to chemical and enzymatic degradation, thus representing valuable alternative surfactants for biotherapeutics.

CMC Regulatory Considerations for Antibody-Drug Conjugates.

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included.

An Intra-Company Analysis of Inherent Particles in Biologicals Shapes the Protein Particle Mitigation Strategy Across Development Stages.

To better understand protein aggregation and inherent particle formation in the biologics pipeline at Novartis, a cross-functional team collected and analyzed historical protein particle issues. Inherent particle occurrences from the past 10 years were systematically captured in a protein particle database. Where the root cause was identified, a number of product attributes (such as development stage, process step, or protein format) were trended. Several key themes were revealed: 1) there was a higher propensity for inherent particle formation with non-mAbs than with mAbs; 2) the majority of particles were detected following manufacturing at scale, and were not predicted by the small-scale studies; 3) most issues were related to visible particles, followed by subvisible particles; 4) 50% of the issues were manufacturing related. These learnings became the foundation of a particle mitigation strategy across development and technical transfer, and resulted in a set of preventive actions. Overall, this study provides further insight into a recognized industry challenge and hopes to inspire the biopharmaceutical industry to transparently share their experiences with inherent particles formation.

Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Technical considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Replacing the Emulsion for Bake-on Siliconization of Containers-Comparison of Emulsion Stability and Container Performance in the Context of Protein Formulations.

Pre-filled syringes have simplified parenteral administration of protein drugs. To ensure an easy and consistent movement of the plunger, the inner glass container surface is typically siliconized. For bake-on siliconization, emulsions are sprayed on and heat treated. Due to the European Union regulation REACh (Regulation concerning theRegistration,Evaluation,Authorisation and Restriction ofChemicals) the use of certain emulsion components, partially constituting the gold standard LiveoTM 365 35% Dimethicone NF Emulsion (LiveoTM 365), becomes restricted and LiveoTM 366 35% Dimethicone NF Emulsion (LiveoTM 366) has been introduced as an alternative. This change may affect the handling properties as well as the silicone layer formed. The purpose of these studies was to identify any differences that may influence the stability and safety of the final drug/device combination product to enable the use of the new emulsion. We compared silicone emulsions LiveoTM 365 and LiveoTM 366 and dilutions focusing on 1) their general physical stability, 2) the thermal degradation process of the emulsions and their components, and 3) the resulting silicone layer concerning chemistry, morphology, and functionality. The results were linked to the assessment of the final product regarding particle formation and short-term stability. A comparison of the emulsions LiveoTM 365 and LiveoTM 366 for bake-on siliconization is presented to support the transition of the latter as it becomes mandatory with REACh. Our studies show that the two emulsions do not significantly differ with respect to handling and stability, the resultant silicone layer characteristics as well as its functionality. We conclude that the transition to the new emulsion will not significantly impact the final product or the layer performance upon storage and with respect to particle formation.

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability - Part 1: Higher molecular weight species and subvisible particle formation.

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 Tg' 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 Tg' in formulations without glass forming sugars. Storage below Tg' did not lead to mAb aggregation. Above Tg', 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 Tg' completely prevents mAb aggregation and particle formation.

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability - part 2: Aggregate formation and oxidation.

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 kD 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.

Computational fluid dynamic simulations of temperature, cryoconcentration, and stress time during large-scale freezing and thawing of monoclonal antibody solutions.

PURPOSE: Large-scale freezing and thawing experiments of monoclonal antibody (mAb) solutions are time and material consuming. Computational Fluid Dynamic (CFD) modeling of temperature, solute composition as well as the stress time, defined as the time between start of freezing and reaching Tg' at any point in the container, could be a promising approach to ease and speed up process development. METHODS: Temperature profiles at six positions were recorded during freezing and thawing of a 2L rectangular bottle and compared to CFD simulations via OpenFOAM. Furthermore, cryoconcentration upon freezing and concentration gradients upon thawing of a mAb solution were predicted and the stress time calculated. RESULTS: Temperature profiles during freezing were accurately matched by the CFD simulation. Thawing time was only 45 min to 60 min longer in the model. The macroscopic cryoconcentration of the mAb was also matched by the simulation; only a highly concentrated region in the top and a diluted core in the geometrical centre of the 2 L bottle were not well reflected in the simulation. The concentration gradient after thawing obtained by simulation as well agreed with the experimental result. In addition, CFD simulations allowed to extract the global temperature distribution, the formation of ice, and thus the distribution of stress in the freezing liquid. CONCLUSION: CFD simulations via OpenFOAM are a promising tool to describe large-scale freezing and thawing of mAb solutions and can help to generate a deeper understanding and to improve testing of the robustness of the processes.

Evaluation of Two Novel Scale-Down Devices for Testing Monoclonal Antibody Aggregation During Large-Scale Freezing.

There is a need for representative small volume devices that reflect monoclonal antibody (mAb) aggregation during freezing and thawing (FT) in large containers. We characterised two novel devices that aim to mimic the stress in rectangular 2 L bottles. The first scale-down device (SDD) consists of a 125 mL bottle surrounded by a 3D printed cover that manipulates heat exchange. The second device, a micro scale-down device (mSDD), adapts cooling and heating of 10 mL vials to extend stress time. MAb aggregation upon repeated FT was evaluated considering formation of higher molecular weight species, subvisible particles, and the increase in hydrodynamic radius, polydispersity index, and optical density at 350 nm. Three different mAb solutions were processed. Both an unshielded 125 mL bottle and the SDD can be used to predict aggregation during FT in 2 L bottles. In specific cases the unshielded 125 mL bottle underestimates whereas the SDD slightly overestimates soluble aggregate formation. The mSDD increases aggregation compared to 10 mL vials but is less representative than the SDD. Ultimately, both SDDs enable characterisation of protein sensitivity to large-scale FT with two orders of magnitude less volume and are superior to simply using smaller bottles.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.