Best Practices for Microbial Challenge In-use Studies to Evaluate the Microbial Growth Potential of Parenteral Biological Products; Industry and Regulatory Considerations.

Microbial challenge in-use studies are performed to evaluate the potential for microbial proliferation in preservative-free single dose biological products after first puncture and potential accidental contamination during dose preparation (e.g. reconstitution, dilution) and storage. These studies, in addition to physicochemical in-use stability assessments, are used as part of product registration to define in-use hold times in Prescribing Information and in the pharmacy manual in the case of clinical products. There are no formal guidance documents describing regulator expectations on how to conduct microbial challenge in-use studies and interpret microbial data to assign in-use storage hold-times. In lieu of guidance, US Food and Drug Administration (FDA) regulators have authored publications and presentations describing regulator expectations. Insufficient or unavailable microbial challenge data can result in shortened in-use hold times, thus microbial challenge data enables flexibility for health care providers (HCPs) and patients, while ensuring patient safety. A cross-industry/FDA in-use microbial working group was formed through the Innovation & Quality (IQ) Consortium to gain alignment among industry practice and regulator expectations. The working group assessed regulatory guidance, current industry practice via a blinded survey of IQ Consortium member companies, and scientific rationale to align on recommendations for experimental design, execution of microbial challenge in-use studies, and a decision tree for microbial data interpretation to assign in-use hold times. Besides the study execution and data interpretation, additional considerations are discussed including use of platform data for clinical stage products, closed system transfer devices (CSTDs), transport of dose solutions, long infusion times, and the use of USP <797> by HCPs for preparing sterile drugs for administration. The recommendations provided in this manuscript will help streamline biological product development, ensure consistency on assignment of in-use hold times in biological product labels across industry, and provide maximum allowable flexibility to HCPs and patients, while ensuring patient safety.

Current Industry Best Practice on in-use Stability and Compatibility Studies for Biological Products.

Evaluating the in-use stability of a biological product including its compatibility with administration components allows to define handling instructions and potential hold times that retain product quality during dose preparation and administration. The intended drug product usage may involve the dilution of drug formulation into admixtures for infusion and exposure to new interfaces of administration components like intravenous (iv) bags, syringes, and tubing. In-use studies assess the potential impact on product quality by simulating drug handling throughout the defined in-use period. Considering the wide range of in-use conditions and administration components available globally, only limited guidance is available from regulators on expected in-use stability data. A working group reviewed and consolidated industry approaches to assess physicochemical stability of traditional protein-based biological products during clinical development and for commercial use. The insights compiled in this review article can be leveraged across the industry and encompass topics such as representative drug product material and administration components, testing conditions, quality attributes evaluated and respective acceptance criteria, applied quality standards, and regulatory requirements. These practices may help companies in the study design, and they may inform discussions with global regulators.

Best Practices and Guidelines (2022) for Scale-up and Technology Transfer in Freeze Drying Based on Case Studies. Part 2: Past Practices, Current Best Practices, and Recommendations.

Scale-up and transfer of lyophilization processes remain very challenging tasks considering the technical challenges and the high cost of the process itself. The challenges in scale-up and transfer were discussed in the first part of this paper and include vial breakage during freezing at commercial scale, cake resistance differences between scales, impact of differences in refrigeration capacities, and geometry on the performance of dryers. The second part of this work discusses successful and unsuccessful practices in scale-up and transfer based on the experience of the authors. Regulatory aspects of scale-up and transfer of lyophilization processes were also outlined including a topic on the equivalency of dryers. Based on an analysis of challenges and a summary of best practices, recommendations on scale-up and transfer of lyophilization processes are given including projections on future directions in this area of the freeze drying field. Recommendations on the choice of residual vacuum in the vials were also provided for a wide range of vial capacities.

Risk Mitigation of Plunger-Stopper Displacement Under Low Atmospheric Pressure by Establishing Design Space for Filling-Stoppering Process of Prefilled Syringes: A Design of Experiment (DoE) Approach.

There is a concern that low atmospheric pressure typically encountered during shipment could result in plunger-stopper displacement in prefilled syringes impacting sterility and container closure integrity (CCI) of drug product.1 In this work, following DoE principles we first investigated the impact of filling and stoppering operating parameters on creation of bubble height as performance parameters among others in nominal 1 mL and 2.25 mL Type I glass prefilled syringes (PFSs) with staked needle and rigid needle shield (RNS). Bubble height ranging from <2.0 mm to >15.0 mm were produced in syringes by filling water and vacuum stoppering at operating vacuum pressure ranging from 400 mbar to 950 mbar using a pilot scale filling-stoppering machine. We found that for a particular nominal fill volume in prefilled syringe, as the stoppering vacuum pressure increased, bubble height decreased resulting in plunger-stopper placed closer to the fill level. Subsequently, syringes with varying bubble size were exposed to reduced atmospheric pressure ranging from 628 Torr to 293 Torr bracketing the low pressure recommended by ASTM D4169 standard to qualify shipping containers for transportation of drug products. We found inverse linear correlation between bubble height and plunger-stopper displacement under low atmospheric pressure. However, plunger-stopper displacement increased exponentially as atmospheric pressure decreased. The results suggest that air bubble size in filled glass syringes should be minimized in order to mitigate sterility and container closure integrity (CCI) risk to drug product in prefilled syringes.

Use of a Predictive Regression Model for Estimating Hold-Up Volume for Biologic Drug Product Presentations.

A drug delivery system is designed to administer a therapeutic dose according to its label claim. Upon delivery of a parenteral drug product, the volume remaining inside the container that cannot be extracted at the end of drug administration is called the hold-up volume (HUV) and is primarily considered product wastage. To meet the label claim, every drug product container is filled with a slight excess volume. For early-stage products in clinical phase, for which material availability is often a limitation, excess volume in drug product containers has to be determined experimentally using several grams of product. In such scenarios, established models that can predict HUV in primary drug product containers would be valuable for product development. The objective of this study was to determine HUV with 95% confidence intervals across various container closures and drug delivery systems by using aqueous PEG 400 solution mimicking the viscosity of biologic drug products. ISO 2R, 6R, and 10R vials and single-use hypodermic syringes attached to a Luer lock needle (25 gauge, 1½ in.) were used to mimic parenteral drug product container and delivery systems for determination of HUV. Glass prefilled syringes in 1 mL and 2.25 mL configurations were also used to determine HUV with 95% confidence intervals. A linear regression model was developed for determination of HUV as a function of viscosity and as a function of container closure and a needle-based delivery system. This model predicting HUV was confirmed by using monoclonal antibodies of varying formulations and viscosities for container closure and delivery systems tested in this study. The model provided here can be used to determine HUV for a particular container closure for a drug solution with known viscosity that can subsequently be used to evaluate fill volume specifications and label claim for a dosage form.

Effect of protein cryoconcentration and processing conditions on kinetics of dimer formation for a monoclonal antibody: A case study on bioprocessing.

Monoclonal antibodies (mAbs) may be prone to self-association leading to formation of dimers, trimers, or other high molecular weight species during bio-processing. In order to implement appropriate manufacturing control strategies during bio-processing, it is important to understand various real life bio-processing conditions where such self-associations may manifest. One such case study is presented here of increase in dimer content for an mAb during scale-up bio-processing and the approach taken to understand the under-lying mechanism. In this example, a therapeutic mAb demonstrated a consistently higher dimer values (~0.5% higher) in the drug product (DP) during release when compared to the same value measured in the corresponding drug substance (DS) lot. This observation was interesting since the DS was supplied frozen, and the DS and DP share the same formulation composition and therefore investigation of this dimer change was the scope of the characterization study. Variable path length spectroscopy and size exclusion chromatography was used for protein quantification and to monitor %dimer respectively during characterization of fill-finish unit operations. At the start of DP manufacturing process, immediately after thaw of bulk DS, a protein concentration gradient was observed and the concentration ranged from 90 mg/mL (top of container) to 210 mg/mL (bottom of container). The dimerization kinetics in the same DS container was dependent on concentration with higher concentrations demonstrating higher rates of dimerization. After the bulk DS was mixed for further processing, %dimer in purified bulk DS was quantitated to be approximately 1.4% which is identical to levels observed during scale-up manufacturing of DP. After each unit operation, the in-process samples tested for %dimer showed a gradual increase in dimer as a function of time over the next 7 days accumulating to 1.8% dimer at the end of DP manufacturing process. Samples subjected to static incubation at 2-8°C and room temperature (RT; 15-25°C) showed a gradual increase in dimer over the same time frame; however, the rate of increase in dimer at RT was higher compared to samples stored at 2-8°C. The results from this demonstrate two important key findings: self-association kinetics of mAbs could be exacerbated by protein cryoconcentration and temperature conditions during bioprocessing. Since these two parameters are commonly encountered during manufacturing, the proposed mitigation strategy is to ensure homogeneity of the bulk DP during processing. The temperature dependent self-association kinetics of mAb could be mitigated by processing at lower temperature (e.g., 2-8°C) and by storing the finished DP at lower temperature after manufacturing. The results from this study also highlight the criticality of setting slightly wider specifications for DP compared to DS following ICH Q6B guidelines.

"Product on Stopper" in a Lyophilized Drug Product: Cosmetic Defect or a Product Quality Concern?

During manufacturing of a lyophilized drug product, operator errors in product handling during loading of product filled vials onto the lyophilizer can lead to a seemingly cosmetic defect which can impact certain critical quality attributes of finished product. In this study, filling of a formulated monoclonal antibody in vials was performed using a peristaltic pump filling unit, and subsequently, the product was lyophilized. After lyophilization, upon visual inspection, around 40% of vials had cosmetic defect with residual product around stopper of the vial and were categorized as "product on stopper" vials, whereas remaining 60% vials with no cosmetic defect were called "acceptable vials." Both groups of vials from 1 single batch were tested for critical quality attributes including protein concentration (ultraviolet absorbance at 280), residual moisture (Karl Fischer), sterility (membrane filtration), and container closure integrity (CCI) (blue dye ingress). Analysis of protein quality attributes such as aggregation, protein concentration, residual moisture showed no significant difference between vials with "product on stopper" and "acceptable vials." However, CCI of the "product on stopper" vials was compromised due to the presence of product around stopper of the vial. The results from this case study demonstrate the following 2 important findings: (1) that a seemingly cosmetic defect may impact product quality, compromising the integrity of the product and (2) that CCI test method can be used as an orthogonal method to sterility testing to evaluate sterility assurance of the product. The corrective action proposed to mitigate this defect is use of a larger sized vial that can potentially minimize this defect that arises because of product handling errors.

Colloidal Instability Fosters Agglomeration of Subvisible Particles Created by Rupture of Gels of a Monoclonal Antibody Formed at Silicone Oil-Water Interfaces.

In this study, we investigated the effect of ionic strength (1.25-231 mM) on viscoelastic interfacial gels formed by a monoclonal antibody at silicone oil-water interfaces, and the formation of subvisible particles due to rupture of these gels. Rates of gel formation and their elastic moduli did not vary significantly with ionic strength. Likewise, during gel rupture no significant effects of ionic strength were observed on particle formation and aggregation as detected by microflow imaging, resonance mass measurement, and size exclusion chromatography. Subvisible particles formed by mechanical rupturing of the gels agglomerated over time, even during quiescent incubation, due to the colloidal instability of the particles.

Gelation of a monoclonal antibody at the silicone oil-water interface and subsequent rupture of the interfacial gel results in aggregation and particle formation.

The formation of viscoelastic gels by a monoclonal antibody (mAb) at the silicone oil-water interface was studied using interfacial shear rheology. At a concentration of 50 µg/mL, the mAb formed gels in less than 1 h, and the gelation time decreased with increasing protein concentration. To probe the effects of mechanical rupture of the interfacial gel layers, a layer of silicone oil was overlaid on the surface of aqueous solutions of mAb, and the interface was ruptured periodically with a needle. Rupture of the interfacial gel resulted in formation of subvisible particles and substantial losses of mAb monomer, which were detected by microflow imaging and quantified by size-exclusion chromatography, respectively. Resonance mass measurement showed that levels of both protein particles and silicone oil droplets increased as the gel was repeatedly ruptured with a needle. In contrast, in samples wherein the interfacial gels were not ruptured, much lower levels of aggregates and particles were detected. Addition of nonionic surfactants (polysorbate 20 or polysorbate 80) protected against aggregation and protein particle formation, with increased protection seen with increasing surfactant levels, and with the greatest inhibition observed in samples containing polysorbate 80.

Partial unfolding of a monoclonal antibody: role of a single domain in driving protein aggregation.

We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 °C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's CH2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the CH2 domains leads to aggregation.