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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 1 H 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., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

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., (© PDA, Inc. 2019.)

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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.

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.

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., (© PDA, Inc. 2017.)

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., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© PDA, Inc. 2015.)

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., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

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.

A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan(®), is described., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Current compendial dissolution and disintegrating testing is unable to mimic physiological conditions affecting gastric drug release from immediate release dosage forms. In order to obtain more realistic data, a novel test setup was developed that we term a 'dynamic open flow through test apparatus'. It is based on the previously described dissolution stress test device and attempts to simulate the intra-gastric dissolution conditions pertinent to immediate release dosage forms administered under fasting conditions with respect to flow rates, intra-gastric temperature profiles and gastric motility. The concept of the dynamic open flow through test apparatus has been tested using five different types of hard capsules: conventional hard gelatin capsules (HGC), three hypromellose based capsules (Vcaps, Vcaps Plus and DRcaps) and pullulan based capsules (Plantcaps). These were of different sizes but all contained 100mg caffeine in each formulation, adjusted to avoid buoyancy by addition of excipient. When the capsules were stressed in the apparatus under the dynamic flow conditions applying mild pressure simulating gastric motility, release from release from Vcaps Plus, Vcaps and Plantcaps capsules was very well comparable to HGC. Capsules are usually swallowed with cold water and the temperature dependency of release from gelatin was noted as a significant factor, since heat exchange in the stomach is slow., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel bio-relevant in vitro dissolution device was designed to mimic intragastric conditions after food intake paying particular consideration to mechanical aspects: the Fed Stomach Model (FSM). The FSM represents a fully computer-controlled dynamic flow-through system, in which dosage forms are hosted in so-called gastric vessels. Dosage form movement profiles as well as pressures can be simulated in a physiologically relevant manner. This proof-of-concept study aimed at the investigation of the effects of individual parameters and complex test programs on the drug delivery behavior of diclofenac sodium bilayer extended release tablets. Magnetic marker monitoring experiments demonstrated the applicability of the FSM to simulate intragastric movement velocities of solid oral dosage forms equivalent to in vivo data. Dissolution experiments revealed the relevance of all simulated parameters (i.e. pressure, dosage form movement and pump rate). Moreover, three different test scenarios with test programs specific for fundus, antrum and gastric emptying considered the variability of intragastric transit of solid oral dosage forms after food intake and were confirmed to be reasonable. Dissolution rates were low under conditions specific for fundus owing to low shear stresses. In contrast, higher amounts of the drug were released under high stress conditions simulating antral transit and gastric emptying. Concluding, the FSM can be a valuable tool for bio-relevant dissolution testing due to its potential of precise and reproducible simulation of mechanical parameters characteristic for the fed stomach., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Typical platform processes for biopharmaceutical products derived from animal cell lines include a parvovirus filtration unit operation to provide viral safety assurance of the drug product. The industry has adopted this platform unit operation and gained a wider understanding of its performance attributes, leading to the possibility of streamlined approaches to virus clearance validation. Here, the concept of virus validation on a parvovirus-grade filter with a single worst-case model virus is presented. Several lines of evidence, including published literature and Amgen's own data, support the use of a parvovirus, such as mouse minute virus (MMV), as a worst-case model virus to assess virus removal by parvovirus filters. The evidence presented includes a discussion of the design and manufacture of virus filters with a size exclusion mechanism for removal. Next, the characteristics of different model viruses are compared and a risk assessment on the selection of the relevant model viruses for clearance studies is presented. Finally, a comprehensive summary of literature and Amgen data is provided, comparing the clearance of larger viruses against MMV. Together, this analysis provides a strong scientific rationale for the use of a single, worst-case model virus for assessing virus removal by parvovirus filters, which will ultimately allow for more efficient and streamlined viral clearance study designs., Lay Abstract: Demonstrating the virus clearance capability of a purification process is an important aspect of biopharmaceutical process development. A key component of the viral safety of the process is the inclusion of a parvovirus-grade filter as an effective and robust virus removal step. Traditional methodologies for viral clearance studies have been based on a conservative, data-intensive approach, but recent trends in the field of virus clearance and process development show evolution towards streamlined and more efficient study designs that are based on understanding the mechanism of viral clearance by downstream unit operations. The publication of scientific datasets and awareness of the underlying mechanisms involved with these unit operations have fueled this trend. Here, the concept of virus validation on a parvovirus-grade filter using a parvovirus as single, worst-case model virus is presented. Multiple lines of evidence are provided to support this proposal, including a review of published literature and Amgen historical data. The adoption of this approach provides benefits in terms of cost savings for executing viral clearance studies, but it also simplifies the necessary dataset and focuses on only supplying value-added information to demonstrate the viral safety of the process., (© PDA, Inc. 2014.)

Studying the effects of drugs on the metabolome constitutes a huge part of the metabolomics discipline. Whether the approach is associated with drug discovery (altered pathways due to the disease that provide future targets and information into the mechanism of action or resistance, etc.) or pharmacometabolomics (studying the outcome of treatment), there have been many aspiring published articles in this area. With specific experimental design, including fingerprinting analysis with different analytical platforms in a non-targeted way, the approach is advancing towards the discovery of markers for the implication of personalised medicine, while also providing information that could help to improve the efficacy and reduce the side effects associated with a treatment. In this review, the evolution of pharmacometabolomics from other areas of drug efficacy metabolomics studies is explored.

An optical immunosensor was developed and validated on the surface of microparticles for the determination of a biopharmaceutical protein. The recombinant human myelin basic protein (rhMBP) was produced in milk of transgenic cows as a His-tagged fusion protein. Previous work indicated exclusive association of rhMBP with milk casein micelles that hindered direct determination of the protein in milk. In this work, a solid phase extraction using a cation exchange matrix was developed in order to liberate rhMBP from casein micelles. A sandwich-type immunoassay was then used for in-process monitoring of the full-length protein in the presence of major milk proteins. The assay was successfully employed for detection of ultra-traces of rhMBP (LOD=6.04 ng mL(-1)≈0.3 n mol L(-1)) and for quantitative determination over a wide concentration range (10.00-10,000.00 ng mL(-1)). The assay was able also to detect the rhMBP in the presence of its human counterpart that lacks the His-tag. The high sensitivity along with the ability of the assay to determine the full length protein enabled monitoring of the stability of rhMBP. The testing protocol is particularly useful for intrinsically unstructured proteins that are extremely sensitive to proteolysis and lack a traceable enzymatic activity. This immunosensor provides a specific, ultrasensitive high throughput tool for in-process monitoring in biopharmaceutical industry., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Recent developments in biopharmaceutical processes twined with a desire to remove cleaning and cross-contamination issues from drug production have led to the widespread introduction of single-use technologies and systems within operations. One key area that end users need to address with the advent of these single-use solutions is the potential for increased levels of extractables and leachables within a process, which need to be evaluated and understood as part of any regulatory submission. A science-based and practical method for characterization of extractables and leachables from single-use systems used in manufacturing antibody-drug conjugates has been developed and described in detail. This risk-based approach minimizes the amount of test work while meeting the regulatory requirements to ensure the drug safety and quality. The test design is optimized and the analytical methods (gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and inductively coupled plasma/mass spectrometry) are shown to be suitable for quantifying and identifying the extracted chemical compounds. Application of this characterization method speeds up the filing process for qualification and validation of single-use systems used in bioprocesses.

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

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.

Agitation of small amounts of liquid is performed routinely in biopharmaceutical process, formulation, and packaging development. Protein degradation commonly results from agitation, but the specific stress responsible or degradation mechanism is usually not well understood. Characterization of the agitation stress methods is critical to identifying protein degradation mechanisms or specific sensitivities. In this study, computational fluid dynamics (CFD) was used to model agitation of 1 mL of fluid by four types of common laboratory agitation instruments, including a rotator, orbital shaker, magnetic stirrer and vortex mixer. Fluid stresses in the bulk liquid and near interfaces were identified, quantified and compared. The vortex mixer provides the most intense stresses overall, while the stir bar system presented locally intense shear proximal to the hydrophobic stir bar surface. The rotator provides gentler fluid stresses, but the air-water interfacial area and surface stresses are relatively high given its low rotational frequency. The orbital shaker provides intermediate-level stresses but with the advantage of a large stable platform for consistent vial-to-vial homogeneity. Selection of experimental agitation methods with targeted types and intensities of stresses can facilitate better understanding of protein degradation mechanisms and predictability for "real world" applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Quality by design (QbD) is a science- and risk-based approach to drug product development. Although pharmaceutical companies have historically used many of the same principles during development, this knowledge was not always formally captured or proactively submitted to regulators. In recent years, the US Food and Drug Administration has also recognized the need for more controls in the drug manufacturing processes, especially for biological therapeutics, and it has recently launched an initiative for Pharmaceutical Quality for the 21st Century to modernize pharmaceutical manufacturing and improve product quality. In the biopharmaceutical world, the QbD efforts have been mainly focused on active pharmaceutical ingredient processes with little emphasis on drug product development. We present a systematic approach to biopharmaceutical drug product development using a monoclonal antibody as an example. The approach presented herein leverages scientific understanding of products and processes, risk assessments, and rational experimental design to deliver processes that are consistent with QbD philosophy without excessive incremental effort. Data generated using these approaches will not only strengthen data packages to support specifications and manufacturing ranges but hopefully simplify implementation of postapproval changes. We anticipate that this approach will positively impact cost for companies, regulatory agencies, and patients, alike., (Copyright © 2011 Wiley-Liss, Inc.)

Single-use biopharmaceutical manufacturing requires monitoring of critical manufacturing parameters. We have developed an approach for passive radio-frequency identification (RFID)-based sensing that converts ubiquitous passive 13.56 MHz RFID tags into inductively coupled sensors. We combine several measured parameters from the resonant sensor antenna with multivariate data analysis and deliver unique capability of multiparameter sensing and rejection of environmental interferences with a single sensor. We demonstrate here the integration of these RFID sensors into single-use biopharmaceutical manufacturing components. We have tested these sensors for over 500 h for measurements of temperature and solution conductivity with the accuracy of 0.1°C (32-48°C range) and accuracy of 0.3-2.9 mS/cm (0.5-230 mS/cm range). We further demonstrate simultaneous temperature and conductivity measurements with an individual RFID sensor with the accuracy of 0.2°C (5-60°C range) and accuracy of 0.9 mS/cm (0.5-183 mS/cm range). Developed RFID sensors provide several important features previously unavailable from other single-use sensing technologies such as the same sensor platform for measurements of physical, chemical, and biological parameters; multi-parameter monitoring with individual sensors; and simultaneous digital identification., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Introduction: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays., Methods: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support., Results: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10μL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less., Discussion: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this study the Nano Spray Dryer B-90 (BÜCHI Labortechnik AG, Flawil, Switzerland) was evaluated with regard to the drying of proteins and the preparation of respirable powders in the size range of 1-5 μm. β-galactosidase was chosen as a model protein and trehalose was added as a stabilizer. The influence of inlet temperature, hole size of the spray cap membrane and ethanol concentration in the spray solution was studied using a 3³ full factorial design. The investigated responses were enzyme activity, particle size, span, yield and shelf life. Furthermore, the particle morphology was examined. The inlet temperature as well as the interaction of inlet temperature and spray cap size significantly influenced the enzyme activity. Full activity was retained with the optimized process. The particle size was affected by the hole size of the spray cap membrane and the ethanol content. The smallest cap led to a monodisperse particle size distribution and the greatest yield of particles of respirable size. Higher product recovery was achieved with lower inlet temperatures, higher ethanol contents and smaller cap sizes. Particle morphology differed depending on the cap size. The protein exhibited higher storage stability when spray dried without ethanol and when a larger spray cap size was used., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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.

There are a multitude of filter designs and mechanisms utilized within the biopharmaceutical industry. Prefilters are commonly pleated or wound filter fleeces manufactured from melt-blown random fiber matrices. These filters are used to remove a high contaminant content within the fluid. Prefilters have a large band of retention ratings and can be optimized to all necessary applications. The most common application for prefilters is to protect membrane filters, which are tighter and more selective than prefilters. Membrane filters are used to polish or sterilize fluids. These filters need to be integrity testable to assess whether or not they meet the performance criteria. Cross-flow filtration can be utilized with micro- or ultrafiltration membranes. The fluid sweeps over the membrane layer and therefore keeps it unblocked. This mode of filtration also allows diafiltration or concentration of fluid streams. Nanofilters are commonly used as viral removal filters. The most common retention rating of these filters is 20 or 50 nm.

Sterilizing and pre-filters are manufactured in different formats and designs. The criteria for the specific designs are set by the application and the specifications of the filter user. The optimal filter unit or even system requires evaluation, such as flow rate, throughput, unspecific adsorption, steam sterilizability and chemical compatibility. These parameters are commonly tested within a qualification phase, which ensures that an optimal filter design and combination finds its use. If such design investigations are neglected it could be costly in the process scale.

Validation of a sterilizing filtration process is critical since it is impossible with currently available technology to measure the sterility of each filled container; therefore, sterility assurance of the filtered product must be achieved through validation of the filtration process. Validating a pharmaceutical sterile filtration process involves three things: determining the effect of the liquid on the filter, determining the effect of the filter on the liquid, and demonstrating that the filter removes all microorganisms from the liquid under actual processing conditions.

Membrane filtration is used within a multitude of processes ranging from dialysis to desalination processes to sterilizing filtration in the pharmaceutical industry. Membranes, nevertheless, have to have special characteristics and properties to serve such specific applications. Microfiltration membranes are utilized in a large range of membrane polymers and structures, which all have individual production process steps to achieve consistently the same membrane parameters. This chapter discusses membrane polymers and production processes in detail.

Throughput for early discovery drug metabolism studies can be increased with the concomitant acquisition of metabolite screening information and quantitative analysis using ultra-fast gradient chromatographic methods. Typical ultra-fast high-performance liquid chromatography (HPLC) parameters used during early discovery pharmacokinetic (PK) studies, for example, employ full-linear gradients over 1-2 min at very high flow rates (1.5-2 mL/min) on very short HPLC columns (2 x 20 mm). These conditions increase sample throughput by reducing analytical run time without sacrificing chromatographic integrity and may be used to analyze samples generated from a variety of in vitro and in vivo studies. This approach allows acquisition of more information about a lead candidate while maintaining rapid analytical turn-around time. Some examples of this approach are discussed in further detail., (Copyright 2002 John Wiley & Sons, Ltd.)

The clearance pattern of a specific substance is very important to estimate its oral availability. Devices or models that simulate clearance in the mouth are important to study the effects and retention time of foods and drugs. This report describes an efficient device which was assembled with low-cost materials in our laboratory and that can be used to study the clearance of cariogenic substrates, mouthwashes, programmed-release drugs as well as adsorption of drugs to enamel. The device can have up to three chambers with varying minimum and maximum volumes that can be eluted simultaneously at different flows. The simulated swallowed volumes are adjustable and the ratio between the maximum and minimum volumes can be programmed. We also present the results of an evaluation study using the device to determine the clearance of fluoride from a fluoride-containing mouthwash, the clearance of a 1% glucose solution and the programmed release of fluoride from a methacrylate bioadhesive using artificial saliva as eluent.

The use of nonbaffled vessels for mixing applications is becoming common in the biopharmaceutical industry but is not sufficiently well studied. Orientation of the impellers off-centered and/or at an angle is necessary to enhance mixing and eliminate swirling that would result without a baffle in a standard tank. This study focuses on characterizing mixing in vessels with the hydrofoil axial flow impellers mounted off-center at 10 degrees to the vertical. Geometrically similar vessels ranging from 100 to 5000 L working volume were used in this study. Mixing performance was successfully correlated to vessel geometric factors.

Polymeric hydrophilic matrices are widely used for controlled-release preparations. The process of drug release is controlled by matrix swelling or polymer dissolution. It has been shown that the swelling of guar gum is affected by concentration of drug and viscosity grade of the polymer. This study examines the mechanism of behavior of guar gum in a polymer-drug matrix. The swelling action of guar gum, in turn, is controlled by the rate of water uptake into the matrices. An inverse relationship exists between the drug concentration in the gel and matrix swelling. This implies that guar gum swelling is one of the factors affecting drug release. The swelling behavior of guar gum is therefore useful in predicting drug release.

The aim of this study was to evaluate an automated method using the Enhancer Cell and compare the release of the corticosteroid triamcinolone acetonide (TA) from commercial semisolid formulations. The method used a modified USP Apparatus 2 using the Enhancer Cell in 200 ml capacity flasks instead of the standard 900 ml flasks. The additional equipment included an adapter plate to position the flasks in the center, a cover to reduce the receptor phase evaporation and smaller sized (1/4 in.) shaft and collets. All products were evaluated prior to their expiration date. Effects of system variables such as the temperature and composition of the receptor medium, stirring speed, and the choice of membrane on the drug release were evaluated. Statistical analysis was carried out using SAS Ver. 6.07 and the slopes and intercepts (of the cumulative release/unit area versus square root of time plots) were compared. TA release was a linear function of the square root of time (P < or = 0.0001), in accordance with Higuchi's model (r2 > or = 0.9 in most cases). Temperature (32 and 37 degrees C) did not affect the drug release (P > 0.32) but a significantly higher release rate was observed (P < or = 0.0001) at 50 degrees C. Stirring speed (50, 100, 200 rpm) (P > 0.26) and receptor media composition (38 and 76% ethanol) (P > 0.68) did not significantly alter the release rates. Membrane selection (regenerated cellulose, polyethylene, and rat skin) was found to be a significant variable (P < or = 0.004). This study demonstrates the use of the Enhancer Cell as an automated quality control tool in the in vitro release testing procedure for semisolid drug formulations.