Delivery Considerations of Highly Viscous Polymeric Fluids Mimicking Concentrated Biopharmaceuticals: Assessment of Injectability via Measurement of Total Work Done "WT".

An account is given of the recent development of the highly viscous complex biopharmaceuticals in relation to syringeability and injectability. The specific objective of this study is to establish a convenient method to examine problem of the injectability for the needle-syringe-formulation system when complex formulations with diverse viscosities are used. This work presents the inter-relationship between needle size, syringe volume, viscosity, and injectability of polymeric solutions having typical viscosities encountered in concentrated biologics, by applying a constant probe crosshead speed on the plunger-syringe needle assembly and continuously recording the force-distance profiles. A computerized texture analyzer was used to accurately capture, display, and store force, displacement, and time data. The force-distance curve and area under the curve are determined, and total work done for complete extrusion of the syringe content was calculated automatically by applying an established Matlab program. Various concentrations (i.e., 0.5-4% w/v of polymeric fluids/dispersions) of polyethylene oxide (PEO) and hydroxypropyl methylcellulose (HPMC) with viscosity ranges of 5-100 cP mimicking concentrated monoclonal antibody solutions and complex biopharmaceutical formulations are investigated. Results indicate that calculated values of total work done to completely extrude the syringe content are the most appropriate parameter that describes viscosity-injection force of dispersed formulations. Additionally, the rheological properties of HPMC and PEO fluids in the context of syringeability and injectability are discussed.

Validation of Dissolution Testing with Biorelevant Media: An OrBiTo Study.

Dissolution testing with biorelevant media has become widespread in the pharmaceutical industry as a means of better understanding how drugs and formulations behave in the gastrointestinal tract. Until now, however, there have been few attempts to gauge the reproducibility of results obtained with these methods. The aim of this study was to determine the interlaboratory reproducibility of biorelevant dissolution testing, using the paddle apparatus (USP 2). Thirteen industrial and three academic laboratories participated in this study. All laboratories were provided with standard protocols for running the tests: dissolution in FaSSGF to simulate release in the stomach, dissolution in a single intestinal medium, FaSSIF, to simulate release in the small intestine, and a "transfer" (two-stage) protocol to simulate the concentration profile when conditions are changed from the gastric to the intestinal environment. The test products chosen were commercially available ibuprofen tablets and zafirlukast tablets. The biorelevant dissolution tests showed a high degree of reproducibility among the participating laboratories, even though several different batches of the commercially available medium preparation powder were used. Likewise, results were almost identicalbetween the commercial biorelevant media and those produced in-house. Comparing results to previous ring studies, including those performed with USP calibrator tablets or commercially available pharmaceutical products in a single medium, the results for the biorelevant studies were highly reproducible on an interlaboratory basis. Interlaboratory reproducibility with the two-stage test was also acceptable, although the variability was somewhat greater than with the single medium tests. Biorelevant dissolution testing is highly reproducible among laboratories and can be relied upon for cross-laboratory comparisons.

Gastrointestinal and Systemic Disposition of Diclofenac under Fasted and Fed State Conditions Supporting the Evaluation of in Vitro Predictive Tools.

This study aimed to gain further insight into the gastrointestinal disposition of the weakly acidic BCS class II drug diclofenac and the implications for systemic drug exposure in humans under fasted and fed state conditions. For this purpose, gastrointestinal and blood samples were collected from healthy volunteers after oral intake of a commercially available tablet of the potassium salt of diclofenac (i.e., Cataflam) in different prandial states. Subsequently, these in vivo data served as a reference for the evaluation of in vitro tools with different levels of complexity, i.e., a conventional USP II dissolution apparatus, a modified version of the dynamic open flow through test apparatus, and the TNO gastrointestinal model equipped with the recently developed advanced gastric compartment (TIMagc). In vivo data suggested impaired drug dissolution and/or immediate precipitation in the fasted stomach, linked to the acidity of the gastric environment. Similarly, a vast presence of solid drug material in the stomach was observed under fed state conditions, which could be attributed to a marked delay in intragastric tablet disintegration after drug intake with a meal. Emptying of solid drug from the stomach into the duodenum generally resulted in rapid intestinal drug (re)dissolution in both test conditions, explaining the absence of a food effect on the extent of overall systemic exposure for diclofenac. In vitro tools were found to be capable of predicting in vivo intraluminal (and systemic) disposition of this compound, the extent of which depended on the degree to which the dynamic nature of the gastrointestinal process(es) to be investigated was simulated.

Effect of Coadministered Water on the In Vivo Performance of Oral Formulations Containing N-Acetylcysteine: An In Vitro Approach Using the Dynamic Open Flow-Through Test Apparatus.

The drug plasma profile after oral administration of immediate release dosage forms can be affected by the human gastrointestinal physiology, the formulation, and the drug itself. In this work, we investigated the in vivo and in vitro performance of two formulations (granules vs. tablet) containing the highly soluble drug N-Acetylcysteine (BCS class I). Thereby, special attention was paid to the effect of the dosage form and the coadministration of water on drug release. Interestingly, the in vivo results from a pharmacokinetic study with 11 healthy volunteers indicated that the drug plasma concentrations were comparable for the tablet given with water as well as for the granules given with and without water. In order to mechanistically understand this outcome, we used a biorelevant dissolution test device, the dynamic open flow-through test apparatus. With the aid of this test apparatus, we were able to simulate biorelevant parameters, such as gastric emptying, hydrodynamic flow as well as physical stress. By this, it was possible to mimic the intake conditions of the clinical trial (i.e., drug intake with and without water). Whereas the experiments in the USP paddle apparatus revealed differences between the two formulations, we could not observe significant differences in the release profiles of the two formulations by using the dynamic open flow-through test apparatus. Even by considering the different intake conditions, drug release was slow and amounted to around 30% until simulated gastric emptying. These results suggest that dissolution was irrespective of coadministered water and the formulation. Despite the high aqueous solubility of N-Acetylcysteine, the limiting factor for drug release was the slow dissolution rate in relation to the gastric emptying rate under simulated gastric conditions. Thus, in case of administration together with water, large amounts of the drug are still present in the stomach even after complete gastric emptying of the water. Consequently, the absorption of the drug is largely controlled by the nature of gastric emptying of the remaining drug. The data of this study indicated that the water emptying kinetics are only determining drug absorption if drug release is rapid enough. If this is not the case, physiological mechanisms, such as the migrating motor complex, play an important role for oral drug delivery.

Calibration of a digital in-line holographic microscopy system: depth of focus and bioprocess analysis.

Digital in-line holographic microscopy (DIHM) allows access to both intensity and phase information with conventional microscopic lateral resolutions. Such imaging techniques can, however, be used to increase the depth of focus compared to conventional compound microscopes. We present a simple DIHM capable of imaging weakly scattering 10 µm diameter microspheres as well as Hs578T cells over a depth of 1 mm; i.e., we demonstrate an increase by a factor of 100 over the depth of focus of a conventional microscope.

Use of artificial digestive systems to investigate the biopharmaceutical factors influencing the survival of probiotic yeast during gastrointestinal transit in humans.

PURPOSE: To evaluate the influence of the main biopharmaceutical factors on the viability of a new probiotic yeast strain, using dynamic in vitro systems simulating human gastric/small intestinal (TIM) and large intestinal (ARCOL) environments. METHODS: The viability of Saccharomyces cerevisiae CNCM I-3856 throughout the artificial digestive tract was determined by microbial counting. We investigated the effects of galenic formulation, food intake, dose, mode and frequency of administration on yeast survival rate. RESULTS: In both fasted and fed states, yeast viability in the upper digestive tract was significantly higher when the probiotic was administered in hydroxypropylmethylcellulose (HPMC) capsules compared to tablets. Food intake led to a delay in yeast release and a two-fold increase in strain survival. Whatever the dose, yeasts were particularly sensitive to the large intestinal environment. High concentrations of probiotic could only be maintained in the colon when it was inoculated twice a day over a 5-h-period. CONCLUSIONS: TIM and ARCOL are complementary in vitro tools relevant for screening purposes, supplying valuable information on the effects of galenic form, food intake and dose regimen on the viability of probiotics throughout the human digestive tract.

In vivo predictive release methods for medicated chewing gums.

Understanding the performance of a drug product in vivo plays a key role in the development of meaningful in vitro drug release methodology. In case of functional chewing gums, the mode and the mechanism of release and the site of application differ significantly from other conventional solid oral dosage forms and require a special consideration to extract meaningful information from clinical studies. In the current study, suitable drug release methodology was developed to predict the in vivo performance of an investigated chewing gum product. Different parameters of the drug release testing apparatus described in the Ph. Eur. and Pharmeuropa were evaluated. Drug release data indicate that the parameters, chewing distance, chewing frequency and twisting motion, affect the drug release. Higher drug release was observed when the frequency was changed from 40 chews/min to 60 chews/min for apparatus A and B, as was the case for the twisting motion when changed from 20º to 40º for apparatus B. As far as the chewing distance is concerned, the release rate was in the following order; apparatus A: 0.3 mm > 0.5 mm > 0.7 mm; apparatus B: 1.4 mm > 1.6 mm > 1.8 mm. A suitable apparatus set-up for in vitro release testing was identified. The method will be useful for the establishment of in vitro in vivo correlations (IVIVC) for medicated chewing gums. Interchangeability of the apparatus for a product is not generally recommended without prior knowledge of the performance of the product, as the construction and principle of operation for the apparatus differ considerably.

Mechanistic analysis of solute transport in an in vitro physiological two-phase dissolution apparatus.

In vitro dissolution methodologies that adequately capture the oral bioperformance of solid dosage forms are critical tools needed to aid formulation development. Such methodologies must encompass important physiological parameters and be designed with drug properties in mind. Two-phase dissolution apparatuses, which contain an aqueous phase in which the drug dissolves (representing the dissolution/solubility component) and an organic phase into which the drug partitions (representing the absorption component), have the potential to provide meaningful predictions of in vivo oral bioperformance for some BCS II, and possibly some BCS IV drug products. Before such an apparatus can be evaluated properly, it is important to understand the kinetics of drug substance partitioning from the aqueous to the organic medium. A mass transport analysis was performed of the kinetics of partitioning of drug substance solutions from the aqueous to the organic phase of a two-phase dissolution apparatus. Major assumptions include pseudo-steady-state conditions, a dilute aqueous solution and diffusion-controlled transport. Input parameters can be measured or estimated a priori. This paper presents the theory and derivation of our analysis, compares it with a recent kinetic approach, and demonstrates its effectiveness in predicting in vitro partitioning profiles of three BCS II weak acids in four different in vitro two-phase dissolution apparatuses. Very importantly, the paper discusses how a two-phase apparatus can be scaled to reflect in vivo absorption kinetics and for which drug substances the two-phase dissolution systems may be appropriate tools for measuring oral bioperformance.

A Comparison Between Emerging and Current Biophysical Methods for the Assessment of Higher-Order Structure of Biopharmaceuticals.

The higher-order structure (HOS) of protein therapeutics is a critical quality attribute directly related to their function. Traditionally, the HOS of protein therapeutics has been characterized by methods with low to medium structural resolution such as Fourier-transform infrared (FTIR), circular dichroism (CD), and intrinsic fluorescence spectroscopy, and differential scanning calorimetry (DSC). Recently, high-resolution nuclear magnetic resonance (NMR) methods have emerged as powerful tools for HOS characterization. NMR is a multi-attribute method with unique capabilities to provide information about all the structural levels of proteins in solution. We have in this study compared 1 D 1H Profile NMR with the established biophysical methods for HOS assessments using a set of blended samples of the monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that Profile NMR can distinguish between most sample combinations (93%), DSC can differentiate 61% of the sample combinations, and near-ultraviolet CD spectroscopy can differentiate 52% of the sample combinations, whereas no significant distinction could be made between any samples using FTIR or intrinsic fluorescence. Our data therefore show that NMR has superior ability to address differences in HOS, a feature that could be directly applicable in comparability and similarity assessments.

Extractables from integrated single-use systems in biopharmaceutical manufacturing. Part I. Study on components (Pall Kleenpak connector and Kleenpak filter capsule).

Single-use systems for manufacturing biopharmaceuticals can include filter capsules, connectors, tubing, and polymeric film biocontainers. In order to tackle the variety of extractable compounds from these fairly complex systems, we first studied such systems' representative components, and then examined an entire single-use system comprised of filtesr, connectors, tubing, and biocontainers. This approach greatly simplifies the identification of the extractable compounds from the whole system. The test design was based on common, actual processes conducted under worst-case conditions. Part 1 of this series of papers describes a systematic study of extractables from two components, a sterile connector and a capsule filter, in water and ethanol as model solvent extractants. The complete extractables results were obtained using a combination of qualified analytical methods. The results indicated that the potential for the connector and the capsule filter to release leachable materials in significant amounts into the chemically compatible drug product is very low, taking into account the less vigorous conditions in most processes and dilution effects when compared to the water and ethanol extraction conditions reported here. Application of study results is discussed.

Larger Pore Size Hollow Fiber Membranes as a Solution to the Product Retention Issue in Filtration-Based Perfusion Bioreactors.

Tangential flow filtration (TFF) and alternating tangential flow (ATF) filtration technologies using hollow fiber membranes are commonly utilized in perfusion cell culture for the production of monoclonal antibodies; however, product retention remains a known and common problem with these systems. To address this issue, commercially available hollow fibers ranging from several hundred kilo-Daltons (kDa) to 0.65 µm in nominal pore size are tested and are all demonstrated to undergo moderate to severe product retention. Further investigation revealed accumulation of particles in the same size range (≈20-200 nm) as the pores. Based on the assumption that these particles contribute to product retention and membrane plugging, a hollow fiber with an unconventionally larger pore size is subsequently identified and demonstrated to drastically reduce product retention with no impact to cell clarification. Furthermore, these hollow fibers demonstrate surprisingly high membrane capacities, making them an attractive solution to the problem of product retention in perfusion reactors.

Enabling Robust and Rapid Raw Material Identification and Release by Handheld Raman Spectroscopy.

A fast, reproducible, non-destructive method to confirm raw material identification in real-time upon material receipt within a warehouse environment is desired. Current practices in pharmaceutical manufacturing often employ compendia methods for raw material identification tests, which require sample preparation prior to time-consuming chemical analysis and often employ subjective spectral comparisons. We have developed, qualified, and validated a rapid objective identity method ("Rapid ID") by Raman spectroscopy using the Bruker BRAVO handheld Raman spectrometer for 46 common raw materials used in upstream and downstream biopharmaceutical cell culture-based processes. Materials in the Raman identification library include amino acids and other solid neat organic chemicals, liquid organics, polyatomic salts, polymers, emulsifiers, peptides, aqueous solutions, and buffers. Selection of reference spectra and hit quality index limit(s) was based upon a comprehensive spectral survey across multiple suppliers and lots to account for normal cause spectral variation. Method repeatability and reproducibility, selectivity, and robustness against various operational and environmental factors (e.g., instrumental variance, material packaging, and thermal effects) were evaluated. Benefits of a handheld Raman Rapid ID approach include significant reduction of the time for raw material quality release from weeks to minutes, enhanced objectivity, and robust data integrity via autonomous electronic reporting. In addition, routine collection of rich spectroscopic data on raw materials can be leveraged to support further continuous improvement initiatives, including routine monitoring of method performance, continuous improvement of the library, proactive detection of shifts in raw material properties, and provision of data for investigations focused on raw materials. Rapid ID methods are consistent with the move toward the principles of Pharma 4.0-high automated processes with continuous process verification and a holistic control strategy.LAY ABSTRACT: A fast, reproducible, non-destructive method is desired to confirm raw material identification in real time upon receipt within a warehouse environment. We have developed, qualified and validated a rapid objective identity method ("Rapid ID") by Raman spectroscopy using the Bruker BRAVO handheld Raman spectrometer for 46 common raw materials used in upstream and downstream biopharmaceutical cell culture-based processes. Benefits of a handheld Raman Rapid ID approach include significant time reduction of raw material quality release from weeks to minutes, enhanced objectivity, and robust data integrity via autonomous electronic reporting. Rapid ID methods are consistent with the move toward the principles of Pharma 4.0: high automated processes with continuous process verification and a holistic control strategy.

Moss bioreactors producing improved biopharmaceuticals.

Plants may serve as superior production systems for complex recombinant pharmaceuticals. Current strategies for improving plant-based systems include the development of large-scale production facilities as well as the optimisation of protein modifications. While post-translational modifications of plant proteins generally resemble those of mammalian proteins, certain plant-specific protein-linked sugars are immunogenic in humans, a fact that restricts the use of plants in biopharmaceutical production so far. The moss Physcomitrella patens was developed as a contained tissue culture system for recombinant protein production in photo-bioreactors. By targeted gene replacements, moss strains were created with non-immunogenic humanised glycan patterns. These were proven to be superior to currently used mammalian cell lines in producing antibodies with enhanced effectiveness.

Review of centrifugal liquid-liquid chromatography using aqueous two-phase solvent systems: its scale-up and prospects for the future production of high-value biologicals.

Future challenges in the field of bioprocessing include developing new downstream processes for the purification and manufacture of protein-based medicines to relieve the predicted bottleneck that may occur as a result of increasingly high titers obtained from fermentation processes. This review considers recent developments in centrifugal liquid-liquid partition chromatography using aqueous two-phase solvent systems, a gentle host medium for biologicals, and the prospect for scale-up and eventual manufacture of high-value pharmaceutical products.

Throughput Optimization of Continuous Biopharmaceutical Manufacturing Facilities.

In order to operate profitably under different product demand scenarios, biopharmaceutical companies must design their facilities with mass output flexibility in mind. Traditional biologics manufacturing technologies pose operational challenges in this regard due to their high costs and slow equipment turnaround times, restricting the types of products and mass quantities that can be processed. Modern plant design, however, has facilitated the development of lean and efficient bioprocessing facilities through footprint reduction and adoption of disposable and continuous manufacturing technologies. These development efforts have proven to be crucial in seeking to drastically reduce the high costs typically associated with the manufacturing of recombinant proteins. In this work, mathematical modeling is used to optimize annual production schedules for a single-product commercial facility operating with a continuous upstream and discrete batch downstream platform. Utilizing cell culture duration and volumetric productivity as process variables in the model, and annual plant throughput as the optimization objective, 3-D surface plots are created to understand the effect of process and facility design on expected mass output. The model shows that once a plant has been fully debottlenecked it is capable of processing well over a metric ton of product per year. Moreover, the analysis helped to uncover a major limiting constraint on plant performance, the stability of the neutralized viral inactivated pool, which may indicate that this should be a focus of attention during future process development efforts.LAY ABSTRACT: Biopharmaceutical process modeling can be used to design and optimize manufacturing facilities and help companies achieve a predetermined set of goals. One way to perform optimization is by making the most efficient use of process equipment in order to minimize the expenditure of capital, labor and plant resources. To that end, this paper introduces a novel mathematical algorithm used to determine the most optimal equipment scheduling configuration that maximizes the mass output for a facility producing a single product. The paper also illustrates how different scheduling arrangements can have a profound impact on the availability of plant resources, and identifies limiting constraints on the plant design. In addition, simulation data is presented using visualization techniques that aid in the interpretation of the scientific concepts discussed.

Trend analysis of performance parameters of pre-packed columns for protein chromatography over a time span of ten years.

Pre-packed small scale chromatography columns are increasingly used for process development, for determination of design space in bioprocess development, and for post-licence process verifications. The packing quality of 30,000 pre-packed columns delivered to customers over a period 10 years has been analyzed by advanced statistical tools. First, the data were extracted and checked for inconsistencies, and then were tabulated and made ready for statistical processing using the programming language Perl (https://www.perl.org/) and the statistical computing environment R (https://www.r-project.org/). Reduced HETP and asymmetry were plotted over time to obtain a trend of packing quality over 10 years. The obtained data were used as a visualized coefficient of variation analysis (VCVA), a process that has often been applied in other industries such as semiconductor manufacturing. A typical fluctuation of reduced HETP was seen. A Tsunami effect in manufacturing, the effect of propagation of manufacturing deviations leading to out-of-specification products, was not observed with these pre-packed columns. Principal component analysis (PCA) showed that all packing materials cluster. Our data analysis showed that the current commercially available chromatography media used for biopharmaceutical manufacturing can be reproducibly and uniformly packed in polymer-based chromatography columns, which are designed for ready-to-use purposes. Although the number of packed columns has quadrupled over one decade the packing quality has remained stable.

A scale-down cross-flow filtration technology for biopharmaceuticals and the associated theory.

Use of microfiltration (MF) and ultrafiltration (UF) in cross-flow mode has been intensifying in downstream processing for expensive biopharmaceuticals. A scale-down cross-flow module with ring channel was constructed for reducing costs and increasing throughput. Commensurate with its validation, a new scale down (or scale up) theoretical framework has been further developed to 3 operational parities: (1) ratio of initial sample volume to membrane area, (2) shear force adjacent to membrane surface, and (3) initial permeate flux. By keeping identical initial physicochemical properties, we show that these 3 operational parities are equivalent to 2 further time-dependent theoretical parities for flux and transmission respectively. Importantly, transmission sensitively reflects membrane conditions for partially transmissible molecules or particles. Computational fluid dynamics simulation was conducted to confirm nearly identical shear forces for the mini and its reference filters. Permeate fluxes in suspension containing Escherichia coli phage T7, a monoclonal antibody (MAb) or other proteins, and transmission (with phage T7) were measured. For application demonstration, diafiltration and concentration modes were applied to the MAb, and separation mode to a mixture of bovine serum albumin and lysozyme. In conclusion, the developed scale-down filter has been shown to behave identically or similarly to its reference filter.

Factors to Govern Soluble and Insoluble Aggregate-formation in Monoclonal Antibodies.

The aggregation formation of monoclonal antibodies as biopharmaceuticals induced by heat stress was evaluated by size-exclusion chromatography, and the formation rate was correlated with several physicochemical parameters of the antibodies to clarify the factors to govern the aggregate formation. The parameters we studied were: the melting temperature (Tm) and the standard enthalpy of the melting point (ΔmH°) evaluated by differential scanning calorimetry under given and common conditions; the wavelength (λmax) and the intensity (Fint) of the maximum fluorescence peak of 1-anilinonaphthalene-8-sulfonate as a probe dye; the z-average diameter (D) evaluated by dynamic light scattering; and the isoelectric point (pI) and the hydrophobic index (Hpho) of the complementarity determining region calculated from the amino acid sequence. Multivariate statistical analysis with these explanatory variables based on Akaike's information criterion indicates that the soluble aggregate formation is negatively correlated with Tm and pI, while the insoluble aggregate formation is positively correlated with Fint and pI. Based on these results, the mechanisms of the aggregate formation and methods to prevent the formation are discussed.

Particle shedding from peristaltic pump tubing in biopharmaceutical drug product manufacturing.

In a typical manufacturing setup for biopharmaceutical drug products, the fill and dosing pump is placed after the final sterile filtration unit in order to ensure adequate dispensing accuracy and avoid backpressure peaks. Given the sensitivity of protein molecules, peristaltic pumps are often preferred over piston pumps. However, particles may be shed from the silicone tubing employed. In this study, particle shedding and a potential turbidity increase during peristaltic pumping of water and buffer were investigated using three types of commercially available silicone tubing. In the recirculates, mainly particles of around 200 nm next to a very small fraction of particles in the lower micrometer range were found. Using 3D laser scanning microscopy, surface roughness of the inner tubing surface was found to be a determining factor for particle shedding from silicone tubing. As the propensity toward particle shedding varied between tubing types and also cannot be concluded from manufacturer's specifications, individual testing with the presented methods is recommended during tubing qualification. Choosing low abrasive tubing can help to further minimize the very low particle counts to be expected in pharmaceutical drug products.

A Review of the Aging Process and Facilities Topic.

Aging facilities have become a concern in the pharmaceutical and biopharmaceutical manufacturing industry, so much that task forces are formed by trade organizations to address the topic. Too often, examples of aging or obsolete equipment, unit operations, processes, or entire facilities have been encountered. Major contributors to this outcome are the failure to invest in new equipment, disregarding appropriate maintenance activities, and neglecting the implementation of modern technologies. In some cases, a production process is insufficiently modified to manufacture a new product in an existing process that was used to produce a phased-out product. In other instances, manufacturers expanded the facility or processes to fulfill increasing demand and the scaling occurred in a non-uniform manner, which led to non-optimal results. Regulatory hurdles of post-approval changes in the process may thwart companies' efforts to implement new technologies. As an example, some changes have required 4 years to gain global approval. This paper will address cases of aging processes and facilities aside from modernizing options.