Evaluating end-to-end continuous antibody manufacture with column-free capture alternatives from economic, environmental, and robustness perspectives.

Process intensification efforts have renewed interest in the potential of end-to-end continuous manufacture with column-free capture alternatives. This article describes a decisional tool that encompasses mass balance and design equations, process economics, stochastic simulation and multi-criteria decision-making and enables the evaluation of different batch, and continuous flowsheets for monoclonal antibody (mAb) manufacture. The traditional batch process was compared with end-to-end continuous bioprocesses with either protein A capture or column-free capture employing aqueous two-phase extraction or precipitation from economic, environmental, and robustness perspectives. The cost of goods analysis predicted that continuous flowsheets could offer substantial cost savings (~20%-40%) over the batch process at low and medium annual commercial demands (100-500 kg); however, at tonnage demands they resulted in either comparable or higher costs. Comparing the continuous options, the continuous flowsheets with protein A or precipitation yielded similar COG/g values, while aqueous two-phase extraction presented higher costs. The analysis of overall process mass intensities accounting for water and consumables suggested that the continuous flowsheet with protein A would result in the lowest environmental burden. When the economic, environmental, and operational criteria were reconciled using multi-criteria decision-making analysis, the continuous protein A-based flowsheet was found to be the most favorable. A target analysis highlighted the need for process improvements in the following parameters to reduce the manufacturing costs of the continuous column-free capture options below that of protein A: the perfusion volumetric productivity, the harvested cell culture fluid percentage in column-free operations, the column-free step yields along with the implementation of buffer concentrates.

A common framework for integrated and continuous biomanufacturing.

There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs. The framework is capable of supporting the manufacturing of most major biopharmaceutical ICB and legacy processes without major changes in the regulatory license. This article reviews the ICB that are being used, or are soon to be used, in a GMP manufacturing setting for recombinant protein production from mammalian cells. The adaptation of the various ICB modes to the common ICB framework will be discussed, along with the pros and cons of such adaptation. The equipment used in the common framework is generally described. This review is presented in sufficient detail to enable discussions of IBC implementation strategy in biopharmaceutical companies and contract manufacturers, and to provide a road map for vendors equipment design. An example plant built on the common framework will be discussed. The flexibility of the plant is demonstrated with batches as small as 0.5 kg or as large as 500 kg. The yearly output of the plant is as much as 8 tons.

Enhanced filtration performance using feed-and-bleed configuration for purification of antibody precipitates.

Precipitation can be used for the initial purification of monoclonal antibodies (mAbs), with the soluble host cell proteins removed in the permeate by tangential flow microfiltration. The objective of this study was to examine the use of a feed-and-bleed configuration to increase the effective conversion (ratio of permeate to feed flow rates) in the hollow fiber module to enable more effective washing of the precipitate. Experiments were performed using human serum Immunoglobulin G (IgG) precipitates formed with 10 mM zinc chloride and 7 wt% polyethylene glycol. The critical flux was evaluated as a function of the shear rate and IgG concentration, with the resulting correlation used to predict conditions that can achieve 90% conversion in a single pass with minimal fouling. Experimental data for both the start-up and steady-state performance are in good agreement with model calculations. These results were used to analyze the performance of an enhanced continuous precipitation-microfiltration process using the feed-and-bleed configuration for the initial capture / purification of a mAb product.

High throughput solubility and redissolution screening for antibody purification via combined PEG and zinc chloride precipitation.

As upstream product titers increase, the downstream chromatographic capture step has become a significant "downstream bottleneck." Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2 ) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2 to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7-7.5 wt/vol% together with 10 mM ZnCl2 gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Feasibility of spectral pH measurement during the low-pH virus inactivation step of continuous therapeutic antibody production.

Acidic virus inactivation is commonly used during production of biotherapeutic products to provide virus safety in case of undetected virus contamination. Accurate pH measurement is required to ensure the product pH reaches a virus-inactivating level (typically 3.5-3.7), and a level post-inactivation that is appropriate for later purification steps (typically 5.5-7.5). During batch low-pH inactivation in discrete tanks, potentiometric glass probes are appropriate for measuring pH. During continuous inactivation for 2-3 weeks in an enclosed product stream, probe calibration drift and lag may lead to poor accuracy, and operational difficulties when compensating for drift. Monitoring the spectral response of compounds (indicators) in the product stream whose spectra are pH-sensitive offers a possible alternative way to measure pH without these drawbacks. Such indicators can already exist in the stream (intrinsic) or can be added (extrinsic). Herein are reported studies evaluating the feasibility of both.Promising ultraviolet screening results with the two extrinsics studied, thiamine and ascorbic acid, led to the addition of both to product stream samples titrated to different potentiometric pH values in the 3.3-4.5 range (a representative range encountered during continuous inactivation), and attempts to model pH using sample ultraviolet spectra. One model, based on variability in six spectral attributes, was able to predict pH of an independent sample set within ±0.07 units at the 95% confidence level. Since a typical inactivating pH tolerance is ±0.1 units, the results show that extrinsic indicators potentially can measure inactivation pH with sufficient accuracy. Suggested future steps and an alternative approach are presented.

Effectiveness of video-assisted, self-directed, and peer-guided learning in the acquisition of surgical skills by veterinary students.

OBJECTIVE: To determine the influence of self-directed learning, peer feedback, or expert feedback on suturing technique of novice veterinary student surgeons. STUDY DESIGN: Prospective, blinded, video feedback study. SAMPLE POPULATION: Three groups of surgery naïve veterinary students, two groups of 37 students and one group with 36 students. METHODS: Each student completed three cruciate sutures in SynDaver skin. Student performance was video recorded and scored with a validated pro forma. Students were randomly divided into three groups: (1) students critically evaluated their own performance, (2) students critically evaluated peer's performance, and (3) students received a peer's evaluation. Each student repeated the surgical task and assessed his or her own performance, guided by the pro forma. Each student received a video with individualized feedback from an expert prior to repeating the task. Scores and times were analyzed. Student and expert evaluations were compared. RESULTS: Task composite score, time to completion, and completion rate did not differ between groups. Student self-assessed scores did not correlate with expert scores. Forty-three percent and 62% of students stated that self-feedback and peer feedback, respectively, were acceptable forms of learning, and 96% of students felt expert feedback was superior to both. CONCLUSION: Video-based self-evaluation and peer-assisted learning were as effective as expert feedback after didactic lecture in teaching suturing technique to novice veterinary surgeons. CLINICAL SIGNIFICANCE: Video-based self-evaluation and peer feedback were viable alternative teaching strategies to didactic lecture and expert feedback alone for instructing novice veterinary surgeons.

Continuous precipitation for monoclonal antibody capture using countercurrent washing by microfiltration.

There is renewed interest in the possibility of using precipitation for initial capture of high-value therapeutic proteins as part of an integrated continuous downstream process. Precipitation is greatly facilitated by the high product titers now achieved in most cell culture processes, in sharp contrast to chromatographic processes whose performance is reduced at high titers. The current study used a combination of reversible cross-linking (zinc chloride, ZnCl2 ) and volume exclusion (polyethylene glycol) agents to precipitate a monoclonal antibody product directly from harvested cell culture fluid using a continuous tubular precipitation reactor. The precipitates were then dewatered and continuously washed using tangential flow filtration, with a countercurrent-staged configuration used to reduce the amount of wash buffer required and increase host cell protein removal. Long-term operation was achieved by operating the membrane modules below the critical filtrate flux to avoid fouling. Experimental results demonstrate the feasibility of this fully continuous integrated precipitation process at bench scale, with design calculations used to explore the key factors affecting the performance of this system for initial antibody capture.

Complete Urogenital and Anorectal Duplication in a Dog.

A 10-week-old sexually intact female golden retriever was evaluated for two functional anal openings and a bipartite vulva. Examination revealed haired skin between two separate anatomically smaller anal openings. On rectal palpation, a soft tissue septum (5 cm) with a mucosal surface between the two anal openings was palpated. In addition, circumferential rectal musculature was not appreciated on the ventral aspect. Urogenital evaluation revealed duplication of the vestibule and vagina with a complete centrally located septum extending dorsoventrally. Computed tomography (CT) of the abdomen and pelvis, vaginocystourethrogram, and colonogram were performed. Complete bifurcation of the urinary bladder with duplication of the urethra, cervix, and vaginal canal was noted. Approximately 2 cm from the rectum, there was a similar bifurcation that converged the colon into two rectal portions and separate anal openings. The owner was counseled on the severity of congenital malformations and a high likelihood of aging-related developmental complications in the future. The owner elected humane euthanasia and a necropsy was performed to confirm the malformations.

Components of an Evidence-Based Analytic Rubric for Use in Medical School Admissions.

Attrition from medical school remains a serious cause of concern for the medical education community. Thus, there is a need to improve our ability to select only those candidates who will succeed at medical school from many highly qualified and motivated applicants. This can be achieved, in part, by reducing the reliance on cognitive factors and increasing the use of noncognitive character traits in high-stakes admissions decisions. Herein we describe an analytic rubric that combines research-derived predictors of medical school success to generate a composite score for use in admissions decisions. The analytic rubric as described herein represents a significant step toward evidenced-based admissions that will facilitate a more consistent and transparent qualitative evaluation of medical school applicants beyond their grades and Medical College Admissions Test scores and contribute to a redesigned and improved admissions process.

Understanding the Impact of Omega-3 Rich Diet on the Gut Microbiota.

Background. Recently, the importance of the gut microbiota in the pathogenesis of several disorders has gained clinical interests. Among exogenous factors affecting gut microbiome, diet appears to have the largest effect. Fatty acids, especially omega-3 polyunsaturated, ameliorate a range of several diseases, including cardiometabolic and inflammatory and cancer. Fatty acids associated beneficial effects may be mediated, to an important extent, through changes in gut microbiota composition. We sought to understand the changes of the gut microbiota in response to an omega-3 rich diet. Case Presentation. This case study investigated changes of gut microbiota with an omega-3 rich diet. Fecal samples were collected from a 45-year-old male who consumed 600 mg of omega-3 daily for 14 days. After the intervention, species diversity was decreased, but several butyrate-producing bacteria increased. There was an important decrease in Faecalibacterium prausnitzii and Akkermansia spp. Gut microbiota changes were reverted after the 14-day washout. Conclusion. Some of the health-related benefits of omega-3 may be due, in part, to increases in butyrate-producing bacteria. These findings may shed light on the mechanisms explaining the effects of omega-3 in several chronic diseases and may also serve as an existing foundation for tailoring personalized medical treatments.

High throughput screening of particle conditioning operations: I. System design and method development.

The biotech industry is under increasing pressure to decrease both time to market and development costs. Simultaneously, regulators are expecting increased process understanding. High throughput process development (HTPD) employs small volumes, parallel processing, and high throughput analytics to reduce development costs and speed the development of novel therapeutics. As such, HTPD is increasingly viewed as integral to improving developmental productivity and deepening process understanding. Particle conditioning steps such as precipitation and flocculation may be used to aid the recovery and purification of biological products. In this first part of two articles, we describe an ultra scale-down system (USD) for high throughput particle conditioning (HTPC) composed of off-the-shelf components. The apparatus is comprised of a temperature-controlled microplate with magnetically driven stirrers and integrated with a Tecan liquid handling robot. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. A comprehensive suite of high throughput analytics enables measurement of product titer, product quality, impurity clearance, clarification efficiency, and particle characterization. HTPC at the 1 mL scale was evaluated with fermentation broth containing a vaccine polysaccharide. The response profile was compared with the Pilot-scale performance of a non-geometrically similar, 3 L reactor. An engineering characterization of the reactors and scale-up context examines theoretical considerations for comparing this USD system with larger scale stirred reactors. In the second paper, we will explore application of this system to industrially relevant vaccines and test different scale-up heuristics.

High throughput screening of particle conditioning operations: II. Evaluation of scale-up heuristics with prokaryotically expressed polysaccharide vaccines.

Multivalent polysaccharide conjugate vaccines are typically comprised of several different polysaccharides produced with distinct and complex production processes. Particle conditioning steps, such as precipitation and flocculation, may be used to aid the recovery and purification of such microbial vaccine products. An ultra scale-down approach to purify vaccine polysaccharides at the micro-scale would greatly enhance productivity, robustness, and speed the development of novel conjugate vaccines. In part one of this series, we described a modular and high throughput approach to develop particle conditioning processes (HTPC) for biologicals that combines flocculation, solids removal, and streamlined analytics. In this second part of the series, we applied HTPC to industrially relevant feedstreams comprised of capsular polysaccharides (CPS) from several bacterial species. The scalability of HTPC was evaluated between 0.8 mL and 13 L scales, with several different scaling methodologies examined. Clarification, polysaccharide yield, impurity clearance, and product quality achieved with HTPC were reproducible and comparable with larger scales. Particle sizing was the response with greatest sensitivity to differences in processing scale and enabled the identification of useful scaling rules. Scaling with constant impeller tip speed or power per volume in the impeller swept zone offered the most accurate scale up, with evidence that time integration of these values provided the optimal basis for scaling. The capability to develop a process at the micro-scale combined with evidence-based scaling metrics provide a significant advance for purification process development of vaccine processes. The USD system offers similar opportunities for HTPC of proteins and other complex biological molecules.

Quantitative high throughput analytics to support polysaccharide production process development.

The rapid development of purification processes for polysaccharide vaccines is constrained by a lack of analytical tools current technologies for the measurement of polysaccharide recovery and process-related impurity clearance are complex, time-consuming, and generally not amenable to high throughput process development (HTPD). HTPD is envisioned to be central to the improvement of existing polysaccharide manufacturing processes through the identification of critical process parameters that potentially impact the quality attributes of the vaccine and to the development of de novo processes for clinical candidates, across the spectrum of downstream processing. The availability of a fast and automated analytics platform will expand the scope, robustness, and evolution of Design of Experiment (DOE) studies. This paper details recent advances in improving the speed, throughput, and success of in-process analytics at the micro-scale. Two methods, based on modifications of existing procedures, are described for the rapid measurement of polysaccharide titre in microplates without the need for heating steps. A simplification of a commercial endotoxin assay is also described that features a single measurement at room temperature. These assays, along with existing assays for protein and nucleic acids are qualified for deployment in the high throughput screening of polysaccharide feedstreams. Assay accuracy, precision, robustness, interference, and ease of use are assessed and described. In combination, these assays are capable of measuring the product concentration and impurity profile of a microplate of 96 samples in less than one day. This body of work relies on the evaluation of a combination of commercially available and clinically relevant polysaccharides to ensure maximum versatility and reactivity of the final assay suite. Together, these advancements reduce overall process time by up to 30-fold and significantly reduce sample volume over current practices. The assays help build an analytical foundation to support the advent of HTPD technology for polysaccharide vaccines. It is envisaged that this will lead to an expanded use of Quality by Design (QbD) studies in vaccine process development.

High throughput quantification of capsular polysaccharides for multivalent vaccines using precipitation with a cationic surfactant.

The increasing requirement for multivalent vaccines containing diverse capsular polysaccharides has created an unmet need for a fast and straightforward assay for polysaccharide titer. We describe a novel and robust assay for the quantitation of anionic capsular polysaccharides. The binding of hexadecyltrimethyammonium bromide (Hb) to anionic capsular polysaccharides results in a precipitation reaction wherein the suspension turbidity is proportional to polysaccharide titer. The turbidity can be quickly measured as absorbance across a range of wavelengths that resolve scattering light. Carbohydrates comprised of repeating units of one to seven monosaccharides with phosphodiester groups, uronic acids, and sialic acids all reacted strongly and there does not appear to be specificity with respect to the particular anionic moiety. The assay is compatible with an array of common buffers across a pH range of 3.0-8.75 and with NaCl concentration exceeding 400 mM. Interference from DNA can be eliminated with a short incubation step with DNase. With these treatments, the assay has been employed in samples as complex as fermentation broth. A two-log dynamic range has been established with a mean relative standard deviation less than 10% across this range although inferior performance has been observed in fermentation broth. The precipitation assay enables the rapid quantitation of anionic polysaccharides. The resulting procedure can robustly measure the titer of myriad anionic capsular polysaccharides (CPS) in 96 samples in less than 30 min using low toxicity reagents and routine laboratory equipment. This development will greatly reduce the effort required to measure polysaccharide titer and yield during process development of polysaccharide vaccines.

The effect of protein A cycle number on the performance and lifetime of an anion exchange polishing step.

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak-partitioning anion-exchange chromatography polishing step used in a mAb purification process was characterized through high-throughput screening (HTS) experiments, small-scale experiments including a cycling study performed on qualified scale-down models, and large-scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in-process control parameter for monitoring the performance of the AEX step.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies.

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.

High-throughput screening of chromatographic separations: I. Method development and column modeling.

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.

High-throughput screening of chromatographic separations: II. Hydrophobic interaction.

A high-throughput screen (HTS) was developed to evaluate the selectivity of various hydrophobic interaction chromatography (HIC) resins for separating a mAb from aggregate species. Prior to the resin screen, the solubility of the protein was assessed to determine the allowable HIC operating region by examining 384 combinations of pH, salt, and protein concentration. The resin screen then incorporated 480 batch-binding and elution conditions with eight HIC resins in combination with six salts. The results from the screen were reproducible, and demonstrated quantitative recovery of the mAb and aggregate. The translation of the HTS batch-binding data to lab-scale chromatography columns was tested for four conditions spanning the range of product binding and selectivity. After accounting for the higher number of theoretical plates in the columns, the purity and recovery of the lab-scale column runs agreed with the HTS results demonstrating the predictive power of the filterplate system. The HTS data were further analyzed by the calculation of pertinent thermodynamic parameters such as the partition coefficient, K(P), and the separation factor, alpha. The separation factor was used to rank the purification capabilities of the resin and salt conditions explored.