Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling.

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.

Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation.

PURPOSE: Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause. The purpose of this study was to develop an assay to detect lipolytic activity in biopharmaceutical DS and DP formulations. METHODS: The assay is based on the hydrolysis of the lipase substrate 4-methylumbelliferyl oleate to yield the fluorescent product 4-methylumbelliferone. RESULTS: First, the assay components and their concentrations (buffer salts and pH, solvent and inhibitor Orlistat) were established and optimized using a model lipase (Porcine pancreatic lipase) and cell culture harvest fluid that exhibited lipolytic activity. The assay was then successfully applied and thereby qualified in protein formulations and at lipase concentrations possibly encountered in actual biopharmaceutical DS and DP formulations. CONCLUSION: The lipase assay can be used to detect lipolytic activity in intermediate and final DS, for example during process optimization in downstream purification, to better and specifically reduce the level, or deplete, lipases from HCPs. The assay is also suitable to be applied during root cause investigations related to polysorbate degradation in biopharmaceutical DP.

In Vivo Stability of Therapeutic Proteins.

Significant efforts are made to characterize molecular liabilities and degradation of the drug substance (DS) and drug product (DP) during various product life-cycle stages. The in vivo fate of a therapeutic protein is usually only considered in terms of pharmacokinetics (PKs) and pharmacodynamics (PDs). However, the environment in the human body differs substantially from that of the matrix (formulation) of the DP and may impact on the stability of an injected therapeutic protein. Stabilizing excipients used in protein formulations are expected to undergo more rapid distribution and dissociation in vivo, compared to a protein as a highly charged macromolecule. Thus, in vivo stability may significantly differ from shelf-life stability. In vivo degradation of the therapeutic protein may alter efficacy and/or safety characteristics such as immunogenicity. Studying the stability of a therapeutic protein in the intended body compartment can de-risk drug development in early stages of development by improving the selection of better clinical lead molecules. This review assesses the considerations when aiming to evaluate the in vivo fate of a therapeutic protein by comparing the physiology of relevant human body compartments and assessing their potential implications on the stability of a therapeutic protein. Moreover, we discuss the limitations of current experimental approaches mimicking physiologic conditions, depending on the desired route of administration, such as intravenous (IV), subcutaneous (SC), intravitreal (IVT), or intrathecal (IT) administration(s). New models more closely mimicking the relevant physiologic environment and updated analytical methods are required to understand the in vivo fate of therapeutic proteins.

Correction to: In Vivo Stability of Therapeutic Proteins.

A manuscript version without peer-review revisions was mistakenly processed and published.

Role of Formulation Parameters on Intravitreal Dosing Accuracy Using 1 mL Hypodermic Syringes.

PURPOSE: Evaluation of product viscosity, density and aeration on the dose delivery and accuracy for intravitreal injections with commonly used commercially available hypodermic 1 mL syringes. METHODS: Six commercially available hypodermic 1 mL syringes with different specifications were used for the study. Syringes were filled with the test solutions with different densities and viscosities. Syringes were also subjected to shaking stress to introduce aeration in the test solutions in the presence of different surfactant concentrations with and without high antibody concentration. Target intravitreal volumes of 100 µL, 50 µL and 30 µL were tested to assess dosing accuracy in a controlled simulated administration setup using DIN ISO 11040-4 guidelines and Zwick/Roell Z010 TN instrument. RESULTS: With increasing product viscosity, higher volumes and hence doses were delivered especially for very low volumes like 50 µL and 30 µL. No impact of increasing product density was found on the delivered dose. The presence of surfactants or high protein concentration can lead to aeration, which also negatively affects the dose accuracy and precision. CONCLUSION: Formulation parameters like viscosity can have an impact on dose delivery using hypodermic syringes for intravitreal injections and on the resulting glide force.

Evaluation of Different Quality-Relevant Aspects of Closed System Transfer Devices (CSTDs).

PURPOSE: Health care professionals can be exposed to hazardous drugs such as cytostatics during preparation of drugs for administration. Closed sytem transfer devices (CSTDs) were introduced to provide protection for healthcare professional against unintended exposure to hazardous drugs. The interest in CSTDs has significantly increased after USP <800> monograph was issued. The majority of the studies published so far on CSTDs have focused on their "containment" function. However, other important attributes for CSTDs with potential importance for product quality impact are not yet fully evaluated. METHODS: In the current study, we evaluated four sytems from different suppliers, in combination with different container closure systems (CCS), using solutions of different viscosity and surface tension. The different CSTD / CCS combinations were tested for (a) containment (integrity) using a highly sensitive helium leak test, (b) the force required for mounting the vial adaptor, (c) contribution to visible and subvisible particles as well as (d) the hold-up volume. RESULTS: Results show that the majority of CSTDs may have leaks varying in size, and that some of them generated visible particles due to stopper coring and subvisible particles, both due to silicon oil and particulate contaminations of the Devices. Finally, the holdup volume was up to 1 mL depending on the CSTD type, vial size and solution viscosity. CONCLUSION: These results show that there is a need to evaluate the compatibility of CSTD systems to select the best system for the intended use and that CSTDs may adversely impact product quality and delivered dose.

Use of the permitted daily exposure (PDE) concept for contaminants of intravitreal (IVT) drugs in multipurpose manufacturing facilities.

A toxicological evaluation to determine the product specific permitted daily exposure (PDE) value is an accepted method to determine a safe limit for the carry-over of product residues in multipurpose manufacturing facilities. The PDE calculation for intravitreal (IVT) injection of small and large molecular weight (MW) drugs follows the guiding principles set for systemic administration. However, there are specific differences with respect to the volume administered with IVT administration, pharmacokinetic and pharmacodynamics (PK-PD) parameters and potential for toxicity. In this publication, we have proposed a method to derive PDEIVT in the presence of IVT dose. In the absence of an IVT dose we have a proposed default extrapolationof the systemic PDE for intravenous (IV) administration to the PDEIVT dose by applying a factor of 500 based on comparison of the volume of vitreous humour with the plasma volume, as well as provided examples for PK-PD and toxicity considerations.

Impact of Vial Washing and Depyrogenation on Surface Properties and Delamination Risk of Glass Vials.

PURPOSE: The proper understanding of glass delamination is important to glass manufacturers, pharmaceutical companies, and health authorities to mitigate the occurrence of glass flakes from the vial when in contact with specific drug product solutions. The surface of glass vials is altered during glass cane- and vial forming processes and is exposed to different stress conditions during drug product processing before coming in contact with the drug product solution. In this study, the impact of vial washing and depyrogenation including an evaluation of various residual water volumes on surface properties of glass vials was investigated for a defined set of vials. METHODS: 3D laser scanning microscopy was established as a new method for topographic analysis of curved surfaces of glass vials operating in high-throughput mode. A subset of vials was subsequently exposed to delamination stress testing and both the stressed solution and inner vial surface were analyzed by a panel of conventional and advanced analytical techniques including 3D laser scanning microscopy. RESULTS: The data showed that vial washing and depyrogenation strongly influenced surface properties, in particular those of uncoated vials. Surface characteristics such as pits increased depending on the process conditions, which especially applies to Expansion 33 vials. Even low residual water volumes of 50 µL after vial washing were sufficient to change the surface properties of the glass and weaken the surface in those positions prone to glass delamination. An increase in pits was related to a greater risk for glass delamination. CONCLUSIONS: Vial processing conditions need to be assessed when aiming at minimizing the glass delamination risk during parenteral product storage.

Considerations for the Use of Polysorbates in Biopharmaceuticals.

PURPOSE: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. METHODS: A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. RESULTS: Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. CONCLUSIONS: An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance.

A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model.

Factors Governing the Precision of Subvisible Particle Measurement Methods - A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe.

PURPOSE: The current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance. METHODS: The performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions. RESULTS: The results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results. CONCLUSIONS: The variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control.

Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities.

Although light obscuration is the "gold standard" for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 µm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 µm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements.

Measuring tissue back-pressure--in vivo injection forces during subcutaneous injection.

PURPOSE: Limited information is available on injection forces of parenterals representing the in vivo situation. Scope of the present study was to investigate the contribution of the subcutaneous (sc) tissue layer to injection forces during in vivo injection. METHODS: Göttingen minipigs received injections of isotonic dextran solutions (1-100 mPas) into the plica inguinalis using different injection rates and volumes (0.025-0.2 mL/s and 2.5 vs. 4.5 mL). RESULTS: The contribution of the sc back-pressure to injection forces was found to increase linearly with viscosity and injection rate ranging from 0.6 ± 0.5 N to 1.0 ± 0.4 N (1 mPas), 0.7 ± 0.2 N to 2.4 ± 1.9 N (10 mPas), and 1.8 ± 0.6 N to 4.7 ± 3.3 N (20 mPas) for injection rates of 0.025 to 0.2 mL/s, respectively. Variability increased with viscosity and injection rate. Values are average values from 10 randomized injections. A maximum of 12.9 N was reached for 20 mPas at 0.2 mL/s; 6.9 ± 0.3 N was determined for 100 mPas at 0.025 mL/s. No difference was found between injection volumes of 2.5 and 4.5 mL. The contribution of the tissue was differentiated from the contribution of the injection device and a local temperature effect. This effect was leading to warming of the (equilibrated) sample in the needle, therefore smaller injection forces than expected compensating tissue resistance to some parts. CONCLUSIONS: When estimating injection forces representative for the in vivo situation, the contribution of the tissue has to be considered as well as local warming of the sample in the needle during injection.

Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles--Differential Centrifugation and FACS.

PURPOSE: The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products. METHODS: Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement. RESULTS: Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 µm: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time-their relative advantages and drawbacks. CONCLUSIONS: FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles.

Subvisible (2-100 µm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Directional freezing and thawing of biologics in drug substance bottles.

Biological drug substance (DS) is typically stored frozen to increase stability. However, freezing and thawing (F/T) of DS can impact product quality and therefore F/T processes need to be controlled. Because active F/T systems for DS bottles are lacking, freezing is often performed uncontrolled in conventional freezers, and thawing at ambient temperature or using water baths. In this study, we evaluated a novel device for F/T of DS in bottles, which can be operated in conventional freezers, generating a directed air stream around bottles. We characterized the F/T geometry and process performance in comparison to passive F/T using temperature mapping and analysis of concentration gradients. The device was able to better control the F/T process by inducing directional bottom-up F/T. As a result, it reduced cryo-concentration during freezing as well as ice mound formation. However, freezing with the device was dependent on freezer performance, i.e. prolonged process times in a highly loaded freezer were accompanied by increased cryo-concentrations. Thawing was faster compared to without the device, but had no impact on concentration gradients and was slower compared to thawing in a water bath. High-performance freezers might be required to fully exploit the potential of directional freezing with this device and allow F/T process harmonization and scaling across sites.

A Survey on Handling and Administration of Therapeutic Protein Products in German and Swiss Hospitals.

Protein products in hospitals often have to be compounded before administration to the patient. This may comprise reconstitution of lyophilizates, dilution, storage, and transport. However, the operations for compounding and administration in the hospital may lead to changes in product quality and possibly even impact patient safety. We surveyed healthcare practitioners from three clinical units using a questionnaire and open dialogue to document common procedures and their justification and to document differences in handling procedures. The survey covered dose compounding, transportation, storage and administration. One key observation was that drug vial optimization procedures were used for some products, e.g., use of one single-use vial for several patients. This included the use of spikes and needles or closed system transfer devices (CSTDs). Filters or light protection aids were used only when specified by the manufacturer. A further observation was a different handling of the overfill in pre-filled infusion containers, possibly impacting total dose. Lastly, we documented the complexity of infusion administration setups for administration of multiple drugs. In this case, flushing procedures or the placement and use of filters in the setup vary. Our study has revealed important differences in handling and administration practice. We propose that drug developers and hospitals should collaborate to establish unified handling procedures.

Impact of the Design of Different Infusion Containers on the Dosing Accuracy of a Therapeutic Drug Product.

Residual volumes of infusion solutions vary greatly due to container and dimensional variances. Manufacturers use overfill to compensate, but the exact amounts vary significantly. This variability in overfill - when carrier solutions are used to dilute other parenteral preparations - may lead to variable concentrations and dosing, hence, potential risk for patients. We analyzed the overfill and residual volume of 22 pre-filled infusion containers and evaluated the impact on the (simulated) dosing accuracy of a therapeutic drug product for different handling scenarios. In addition, compendial properties of the diluents (i.e. sub-visible particles, pH, color and opalescence) were assessed. The overfill and residual volume between different containers for the same diluent varied. As container size increased, the relative volume of overfill decreased while the residual volume remained constant. The design and material of the containers (e.g. port systems) defined the residual volume. Different handling scenarios led to differences in dosing accuracy. As a result, no universal approach applicable for all containers can be defined. To ensure the right dose, it is recommended to pre-select the preferred diluent, evaluate fill volumes of carrier solutions, and assess in-use compatibility of the product solution with its diluent in terms of concentration and volume.

Characterization of Silicone from Closed System Transfer Devices and its Migration into Pharmaceutical Drug Products.

Closed System Transfer Devices (CSTDs) are increasingly used in healthcare settings to facilitate compounding of hazardous drugs but increasingly also therapeutic proteins. However, their use may significantly impact the quality of the sterile product. For example, contamination of the product solution may occur by leaching of silicone or particulates from the CSTDs. It was therefore the aim of the present study to identify and quantify the types of silicone oil in a panel of typically used CSTDs. Particles found after simulated CSTD compounding processes were evaluated using Light Obscuration and Micro-Flow Imaging and were confirmed to be silicone oil particles. The number of particulates shed from CTSDs was in single cases exceeding pharmacopeial limits for a final parenteral product. Using X-ray microtomography, lubrication was shown to be primarily applied at connecting parts of the CSTD. Quantitative and qualitative analysis by Fourier transform infrared spectroscopy (FTIR) revealed a total released amount between 0.8 and 16 mg per CSTD of polydimethylsiloxane or polymethyltrifluoropropylsiloxane per CSTD. While pronounced differences in total silicone content between CSTDs were observed, it did not fully correlate with particle contamination in the test solutions, potentially due to variations in CSTD design. The impact of typical surfactants in biological formulations on silicone migration into product was additionally evaluated. We conclude that CSTDs may compromise final product quality, as (different types of) silicone oil may be released from these devices and contaminate the administered product.