Nonclinical Development of Biologics: Integrating Safety, Pharmacokinetics, and Pharmacodynamics to Create Smarter and More Flexible Nonclinical Safety Programs Optimizing Animal Use.

Safety assessment of biological drugs has its challenges due to the multiple new different modalities, for example, antibody-drug conjugates, bispecifics, nanobodies, fusion proteins and advanced therapy medicinal products (ATMPs), their different pharmacokinetic and pharmacodynamic properties, and their ability to trigger immunogenicity and toxicity. In the public and in the pharmaceutical industry, there is a strong and general desire to reduce the number of animals used in research and development of drugs and in particular reducing the use of nonhuman primates. Important discussions and activities are ongoing investigating the smarter designs of early research and dose range finding studies, reuse of animals, and replacing animal experiments with in vitro studies. Other important challenges include absence of a relevant species and design of studies and developing genetically modified animals for special investigative toxicology studies. Then, the learnings and challenges from the development of the first ATMPs are available providing valuable insights in the development path for these new potentially transformative treatments. Finally, development of strategies for assessment of immunogenicity and prediction of translation of immunogenicity and associated findings to the clinic. On this, the eighth meeting for the European BioSafe members, these challenges served as the basis for the presentations and discussions during the meeting. This article serves as the workshop report reviewing the presentations and discussions at the meeting.

Residual Seal Force Testing: A Suitable Method for Seal Quality Determination of (High Potent) Parenterals.

Vial capping plays a critical role in the drug product manufacturing process owing to the complex interplay of several adjustable process steps. Seal quality and integrity and containment assurance are essential for parenteral pharmaceuticals, as the vial's content may be contaminated or, in the case of highly potent drugs (e.g., antibody drug conjugates), may bear a risk of contamination. The residual seal force (RSF) method can enable further insight in capping equipment settings independently of the container closure system (CCS) and their resulting seal quality.The present study investigates the accuracy of the RSF method focusing on different force settings, RSF development over time, distance between capping plates and vial neck (roller-axis), time point of flip-off button removal, and internal and external vial pressure differences (flight simulation and vials closed under vacuum).Results show that the forces used on an RSF tester should be kept low to minimize CCS deformation, and a period of stable RSF values after the initial decrease should be implemented between capping and RSF measurement to increase accuracy. Variations in the distance between the capping plates and vial neck (roller-axis) can result in incomplete crimps or visual defects of the seals. In addition, the time point of flip-off button removal as part of the sample preparation had no significant impact on RSF measurements. Finally, pressure differences between the vial interior and exterior had no significant impact on the RSF data.LAY ABSTRACT: Vial capping plays a critical role in the drug product manufacturing process due to the complex interplay of several adjustable process steps. Seal quality, integrity, and containment are essential for parenteral pharmaceuticals, as the vial's content varies and may be contaminated, sensitive to stress, and/or highly potent (eg, antibody drug conjugates). The residual seal force (RSF) method can enable further insight in capping equipment settings independently of the container closure system and their resulting seal quality.In this study, we determined RSF values by applying different force settings of the RSF tester and investigated the influence of sample preparation on the determination of RSF. Furthermore, the capping process parameter roller-axis was evaluated by RSF and visual inspection. In addition, we investigated the influence of pressure differences of vials on the RSF as they occurred during air transport and products closed under vacuum.

Evaluation of Container Closure System Integrity for Storage of Frozen Drug Products: Impact of Capping Force and Transportation.

Frozen-state storage and cold-chain transport are key operations in the development and commercialization of biopharmaceuticals. Today, several marketed drug products are stored (and/or shipped) under frozen conditions to ensure sufficient stability, particularly for live viral vaccines. When these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial at the target's lowest temperature to ensure container closure integrity and thus both sterility and safety of the drug product. The container closure integrity assessment in the frozen state (e.g., -20°C, -80°C) should include container closure integrity (CCI) of the container closure system (CCS) itself, impact of processing (e.g., capping process on CCI), and impact of shipment and movement on CCI in the frozen state. The objective of this work was to evaluate the impact of processing and shipment on CCI of a CCS in the frozen state. The impact on other quality attributes was not investigated. In this light, the ThermCCI method was applied to evaluate the impact of shipping stress and variable capping force on CCI of frozen vials and to evaluate the temperature limits of rubber stoppers. In conclusion, retaining CCI during cold storage is mostly a function of vial-stopper combination, and temperatures below -40°C may pose a risk to the CCI of a frozen drug product. Variable capping force may have an influence on the CCI of a frozen drug product if not appropriately assessed. Regarding the impact of shipment on the CCI of glass vials, no indication was given at room temperature, -20°C, or -75°C when compared with static storage at such temperatures.LAY ABSTRACT: Today, several marketed products are stored (and/or shipped) under frozen conditions to ensure sufficient stability. When these products are stored in glass vials with stoppers, the elastomer of the stopper needs to be flexible enough to seal the vial and ensure container closure integrity and thus both sterility and safety of the drug product. The impact of processing and shipment on the container closure integrity (CCI) of a container closure system (vial, stopper, and flip-off cap) in the frozen state is assessed. A helium-leakage test at low temperature (ThermCCI) was used to evaluate the impact of shipping stress and variable capping force on CCI of frozen vials as well as the temperature limits of rubber stoppers. In conclusion, it was found that retaining CCI during cold storage is mostly a function of vial-stopper combination and that temperatures below -40°C may pose a risk to the CCI of a frozen drug product. Variable capping force may have an influence on the CCI of a frozen drug product if not appropriately assessed. Additionally, it was observed that the shipment of the frozen glass vials did not affect the CCI.

Biotherapeutics in non-clinical development: Strengthening the interface between safety, pharmacokinetics-pharmacodynamics and manufacturing.

Biological drugs comprise a wide field of different modalities with respect to structure, pharmacokinetics and pharmacological function. Considerable non-clinical experience in the development of proteins (e.g. insulin) and antibodies has been accumulated over the past thirty years. In order to improve the efficacy and the safety of these biotherapeutics, Fc modifications (e.g. Fc silent antibody versions), combinations (antibody-drug conjugates, protein-nanoparticle combinations), and new constructs (darpins, fynomers) have been introduced. In the last decade, advanced therapy medicinal products (ATMPs) in research and development have become a considerable and strongly growing part of the biotherapeutic portfolio. ATMPs consisting of gene and cell therapy modalities or even combinations of them, further expand the level of complexity, which already exists in non-clinical development strategies for biological drugs and has thereby led to a further diversification of expertise in safety and PKPD assessment of biological drugs. It is the fundamental rationale of the BioSafe meetings, held yearly in the EU and in the US, to convene experts on a regular basis and foster knowledge exchange and mutual understanding in this fast growing area. In order to reflect at least partially the variety of the biotherapeutics field, the 2016 EU BioSafe meeting addressed the following topics in six sessions: (i) In vitro Meets in vivo to Leverage Biologics Development (ii) New developments and regulatory considerations in the cell and gene therapy field (iii) CMC Challenges with Biologics development (iv) Minipigs in non-clinical safety assessment (v) Opportunities of PKPD Assessment in Less Common Administration Routes In the breakout sessions the following questions were discussed: (i) Cynomolgus monkey as a reprotoxicology Species: Impact of Immunomodulators on Early Pregnancy Maintenance (ii) Safety Risk of Inflammation and Autoimmunity Induced by Immunomodulators (iii) Experience with non-GMP Material in Pivotal Non-clinical Safety Studies to Support First in Man (FiM) Trials (iv) Safety Assessment of Combination Products for Non-oncology.

Challenges for the pharmaceutical technical development of protein coformulations.

OBJECTIVES: This review discusses challenges to stability, analytics and manufacturing of protein coformulations. Furthermore, general considerations to be taken into account for the pharmaceutical development of coformulated protein drug products are highlighted. KEY FINDINGS: Coformulation of two or more active substances in one single dosage form has recently seen increasing use offering several advantages, such as increased efficacy and/or the overall reduction of adverse event incidents in patients. Most marketed coformulated drug products are composed of small molecules. As proteins are not only comparatively large but also complex molecules, the maintenance of their physicochemical integrity within a formulation throughout pharmaceutical processing, storage, transport, handling and patient administration to ensure proper pharmacokinetics and pharmacodynamics in vivo already represents various challenges for single-entity products. Thus, nowadays, only sparse biologics-based coformulations can be found, as additional complexity during development is given for these products. SUMMARY: The complexity of the dosage form and the protein molecules results into additional challenges to formulation, manufacture, storage, transport, handling and patient administration, stability and analytics during the pharmaceutical development of protein coformulations. Various points have to be considered during different stages of development in order to obtain a safe and efficacious product.

Impact of Vial Capping on Residual Seal Force and Container Closure Integrity.

UNLABELLED: The vial capping process is a critical unit operation during drug product manufacturing, as it could possibly generate cosmetic defects or even affect container closure integrity. Yet there is significant variability in capping equipment and processes, and their relation to potential defects or container closure integrity has not been thoroughly studied. In this study we applied several methods-residual seal force tester, a self-developed system of a piezo force sensor measurement, and computed tomography-to characterize different container closure system combinations that had been sealed using different capping process parameter settings. Additionally, container closure integrity of these samples was measured using helium leakage (physical container closure integrity) and compared to characterization data. The different capping equipment settings lead to residual seal force values from 7 to 115 N. High residual seal force values were achieved with high capping pre-compression force and a short distance between the capping plate and plunge. The choice of container closure system influenced the obtained residual seal force values. The residual seal force tester and piezoelectric measurements showed similar trends. All vials passed physical container closure integrity testing, and no stopper rupture was seen with any of the settings applied, suggesting that container closure integrity was warranted for the studied container closure system with the chosen capping setting ranges. LAY ABSTRACT: The vial capping process is a critical unit operation during drug product manufacturing, as it could possibly generate cosmetic defects or even affect container closure integrity. Yet there is significant variability in capping equipment and processes, and their relation to potential defects or container closure integrity has not been thoroughly studied. In this study we applied several methods-residual seal force tester, a self-developed system of a piezo force sensor measurement, and computed tomography-to characterize different container closure system combinations that had been sealed using different capping process parameter settings. The residual seal force tester can analyze a variety of different container closure systems independent of the capping equipment. An adequate and safe residual seal force range for each container closure system configuration can be established with the residual seal force tester and additional methods like computed tomography scans and leak testing. In the residual seal force range studied, the physical container closure integrity of the container closure system was warranted.

Influence of Different Container Closure Systems and Capping Process Parameters on Product Quality and Container Closure Integrity (CCI) in GMP Drug Product Manufacturing.

Capping equipment used in good manufacturing practice manufacturing features different designs and a variety of adjustable process parameters. The overall capping result is a complex interplay of the different capping process parameters and is insufficiently described in literature. It remains poorly studied how the different capping equipment designs and capping equipment process parameters (e.g., pre-compression force, capping plate height, turntable rotating speed) contribute to the final residual seal force of a sealed container closure system and its relation to container closure integrity and other drug product quality parameters. Stopper compression measured by computer tomography correlated to residual seal force measurements.In our studies, we used different container closure system configurations from different good manufacturing practice drug product fill & finish facilities to investigate the influence of differences in primary packaging, that is, vial size and rubber stopper design on the capping process and the capped drug product. In addition, we compared two large-scale good manufacturing practice manufacturing capping equipment and different capping equipment settings and their impact on product quality and integrity, as determined by residual seal force.The capping plate to plunger distance had a major influence on the obtained residual seal force values of a sealed vial, whereas the capping pre-compression force and the turntable rotation speed showed only a minor influence on the residual seal force of a sealed vial. Capping process parameters could not easily be transferred from capping equipment of different manufacturers. However, the residual seal force tester did provide a valuable tool to compare capping performance of different capping equipment. No vial showed any leakage greater than 10(-8)mbar L/s as measured by a helium mass spectrometry system, suggesting that container closure integrity was warranted in the residual seal force range tested for the tested container closure systems. LAY ABSTRACT: Capping equipment used in good manufacturing practice manufacturing features different designs and a variety of adjustable process parameters. The overall capping result is a complex interplay of the different capping process parameters and is insufficiently described in the literature. It remains poorly studied how the different capping equipment designs and capping equipment process parameters contribute to the final capping result.In this study, we used different container closure system configurations from different good manufacturing process drug product fill & finish facilities to investigate the influence of the vial size and the rubber stopper design on the capping process. In addition, we compared two examples of large-scale good manufacturing process capping equipment and different capping equipment settings and their impact on product quality and integrity, as determined by residual seal force.

The Pharmaceutical Capping Process-Correlation between Residual Seal Force, Torque Moment, and Flip-off Removal Force.

The majority of parenteral drug products are manufactured in glass vials with an elastomeric rubber stopper and a crimp cap. The vial sealing process is a critical process step during fill-and-finish operations, as it defines the seal quality of the final product. Different critical capping process parameters can affect rubber stopper defects, rubber stopper compression, container closure integrity, and also crimp cap quality. A sufficiently high force to remove the flip-off button prior to usage is required to ensure quality of the drug product unit by the flip-off button during storage, transportation, and until opening and use. Therefore, the final product is 100% visually inspected for lose or defective crimp caps, which is subjective as well as time- and labor-intensive. In this study, we sealed several container closure system configurations with different capping equipment settings (with corresponding residual seal force values) to investigate the torque moment required to turn the crimp cap. A correlation between torque moment and residual seal force has been established. The torque moment was found to be influenced by several parameters, including diameter of the vial head, type of rubber stopper (serum or lyophilized) and type of crimp cap (West(®) or Datwyler(®)). In addition, we measured the force required to remove the flip-off button of a sealed container closure system. The capping process had no influence on measured forces; however, it was possible to detect partially crimped vials. In conclusion, a controlled capping process with a defined target residual seal force range leads to a tight crimp cap on a sealed container closure system and can ensure product quality. LAY ABSTRACT: The majority of parenteral drug products are manufactured in a glass vials with an elastomeric rubber stopper and a crimp cap. The vial sealing process is a critical process step during fill-and-finish operations, as it defines the seal quality of the final product. An adequate force to remove the flip-off button prior to usage is required to ensure product quality during storage and transportation until use. In addition, the complete crimp cap needs to be fixed in a tight position on the vial. In this study, we investigated the torque moment required to turn the crimp cap and the force required to remove the flip-off button of container closure system sealed with different capping equipment process parameters (having different residual seal force values).

The pharmaceutical vial capping process: Container closure systems, capping equipment, regulatory framework, and seal quality tests.

Parenteral drug products are protected by appropriate primary packaging to protect against environmental factors, including potential microbial contamination during shelf life duration. The most commonly used CCS configuration for parenteral drug products is the glass vial, sealed with a rubber stopper and an aluminum crimp cap. In combination with an adequately designed and controlled aseptic fill/finish processes, a well-designed and characterized capping process is indispensable to ensure product quality and integrity and to minimize rejections during the manufacturing process. In this review, the health authority requirements and expectations related to container closure system quality and container closure integrity are summarized. The pharmaceutical vial, the rubber stopper, and the crimp cap are described. Different capping techniques are critically compared: The most common capping equipment with a rotating capping plate produces the lowest amount of particle. The strength and challenges of methods to control the capping process are discussed. The residual seal force method can characterize the capping process independent of the used capping equipment or CCS. We analyze the root causes of several cosmetic defects associated with the vial capping process.

New insight into the role of polyethylene glycol acting as protein release modifier in lipidic implants.

PURPOSE: It has recently been shown that the addition of polyethylene glycol 6000 (PEG) to lipidic implants fundamentally affects the resulting protein release kinetics and moreover, the underlying mass transport mechanisms (Herrmann, Winter, Mohl, F. Siepmann, & J. Siepmann, J. Control. Release, 2007). However, it is yet unclear in which way PEG acts. It was the aim of this study to elucidate the effect of PEG in a mechanistic manner. MATERIALS AND METHODS: rh-interferon alpha-2a (IFN-alpha)-loaded, tristearin-based implants containing various amounts of PEG were prepared by compression. Protein and PEG release was monitored in phosphate buffer pH 4.0 and pH 7.4. IFN-alpha solubility and stability were assessed by reverse phase and size exclusion HPLC, SDS PAGE, fluorescence and FTIR. RESULTS: Importantly, in presence of PEG IFN-alpha was drastically precipitated at pH 7.4. In contrast, at pH 4.0 up to a PEG concentration of 20% no precipitation occurred. These fundamental effects of PEG on protein solubility were reflected in the release kinetics of IFN-alpha from the tristearin implants: At pH 7.4 the protein release rates remained nearly constant over prolonged periods of time, whereas at pH 4.0 high initial bursts and continuously decreasing release rates were observed. Interestingly, it could be shown that IFN-alpha release was governed by pure diffusion at pH 4.0, irrespective of the PEG content of the matrices. In contrast, at pH 7.4 both--the limited solubility of the protein as well as diffusion through tortuous liquid-filled pores--are dominating. CONCLUSIONS: For the first time it is shown that the release of pharmaceutical proteins can be controlled by an in-situ precipitation within inert matrices.

Mechanisms controlling protein release from lipidic implants: effects of PEG addition.

Different types of tristearin-based implants for controlled rh-interferon alpha-2a (IFN-alpha) release were prepared by compression and thoroughly characterised in vitro. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was added as a co-lyophilisation agent for protein stabilisation and different amounts of polyethylene glycol (PEG) as efficient protein release modifier. To get deeper insight into the underlying mass transport mechanisms, the release of IFN-alpha, HP-beta-CD and PEG into phosphate buffer pH 7.4 was monitored simultaneously and appropriate analytical solutions of Fick's second law of diffusion were fitted to the experimental results. Importantly, the addition of only 5-20% PEG to the lipidic implants significantly altered the resulting protein release rates and the relative importance of the underlying mass transport mechanisms. The release of IFN-alpha from PEG-free implants was purely diffusion controlled. In contrast, in PEG-containing devices other phenomena were also involved in the control of protein release: the IFN-alpha release rate remained about constant over prolonged periods of time and the total amounts of mobile IFN-alpha increased. Interestingly, the release of PEG itself as well as of HP-beta-CD from the implants remained purely diffusion controlled, irrespective of the amount of added PEG. Thus, different mass transport mechanisms govern the release of the drug, co-lyophilisation agent and release modifier out of the lipidic implants.

NIR spectroscopy-a non-destructive analytical tool for protein quantification within lipid implants.

Lipid implants have been proposed as promising sustained release devices for the parenteral application of pharmaceutical proteins. Near infrared spectroscopy (NIRS) has been reported in the literature to be a non-destructive tool for drug quantification within controlled release matrix systems based on poly-(lactic-co-glycolic) acid (PLGA). The objective of this study was to evaluate the potential application of NIRS for protein content determination within lipid matrices containing stabilizing and release modifying additives. Bovine serum albumin (BSA) and rh-interferon alpha-2a (IFN alpha-2a) were initially lyophilized with trehalose and then blended with tristearin (matrix material) and optionally with polyethylene glygol 6000 (PEG, release modifier). Implants were prepared by compression. NIR transmittance spectra were measured on a NIRTab spectrometer. Partial least squares regression (PLSR) calibration models were developed to predict protein content in implants from the NIRS results. Additional samples were measured after performing release studies. It could be shown that NIRS allowed protein quantification in complex matrix systems with good accuracy after implant manufacture and during release studies [e.g., standard error of prediction (SEP) between 57 microg-176 microg]. In addition, small protein amounts down to 70 microg of incorporated protein per implant could be determined, thus demonstrating the low detection limit of NIRS.

Continuous release of Rh-interferon (alpha-2a from triglyceride implants: storage stability of the dosage forms.

Tristearin implants containing polyethylene glycol 6000 (PEG) were shown to be a promising platform for the delivery of pharmaceutical proteins for periods up to 1 month. The objective of this study was to investigate the storage stability of the lipid devices, as long-term storage stability ensuring acceptable shelf-life can be considered the most important parameter for commercially viable sustained-release dosage forms. Rh-Interferon alpha-2a was stabilized by a lyophilization process using either trehalose or hydroxypropyl-beta-cyclodextrin as stabilizer. Tristearin implants containing the lyophilized protein material and 10% PEG were stored over 3 months and 6 months, both at 4 degrees C and room temperature, before release studies were initiated. Data from stored implants demonstrated trehalose not to be effective to provide full protein stabilization during long-term storage of the lipid matrices, this was apparent from both the reduced total drug level liberated and the release of aggregated specimen compared to the situation immediately after implant manufacture. In contrast, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) exhibited a high potential for protein stabilization within the matrices during both storage and release. Generally, 95% of the incorporated protein was delivered continuously within 1 month in monomeric form, even after 6 months' storage of the implants at room temperature.

Continuous release of rh-interferon alpha-2a from triglyceride matrices.

The use of biodegradable polymeric matrices as controlled release systems is known to be associated with various drawbacks. The objective of this study was to develop an alternative delivery system based on triglycerides, thereby aiming for sustained continuous protein release. Tristearin implants containing lyophilised rh-interferon alpha-2a (IFN-alpha) and varying amounts of polyethylene glycol 6000 (PEG) were prepared by compression. Release studies exhibited that more than 90% of the incorporated IFN-alpha can be liberated in a continuous way over 1 month from systems containing 10% PEG. Integrating hydroxypropyl-beta-cyclodextrin (HP-beta-CD) into the matrices proved to stabilise IFN-alpha and led to a higher and faster protein release due to solubilising effects. The protein was released in virtually monomeric form. Scanning electron microscopy (SEM) and mercurial porosimetry revealed the matrices forming an interconnected pore network.