Structural and functional characteristics of virgin and fouled Protein A MabSelect resin cycled in a monoclonal antibody purification process.

The structural and functional characteristics of the Protein A MabSelect resin are determined for a virgin sample and for samples removed from a column that had been operated in an antibody capture process which had shown losses in product recovery over fewer than 20 cycles. Compared to the virgin resin, the cycled samples show reduced porosity and apparent pore size based on inverse size exclusion chromatography while transmission electron microscopy (TEM) shows accumulation of foulants on the cycled resin. Adsorption isotherms, batch adsorption kinetics, and batch desorption kinetics, obtained using the antibody in purified form, show that the cycled samples have about 10% lower binding capacity and slower mass transfer. Confocal scanning laser microscopy shows, however, that different degrees of fouling exist for different beads in the cycled samples, which may correspond to the existence of areas exposed to minimal or no flow in the process column. Replacing the standard cleaning procedure with an improved multi-step cleaning protocol prevented the accumulation of foulants in the resin beads, as evident from TEM, and resulted in a stable operation with high recovery.

Nature of foulants and fouling mechanism in the Protein A MabSelect resin cycled in a monoclonal antibody purification process.

The composition and origin of foulants and their spatial distribution within the particles of the Protein A MabSelect resin cycled in a mAb purification process are determined using electron and confocal microscopy techniques with gold and fluorescently labeled protein probes that associate with the foulants. The results show that the foulants are primarily related to the mAb product, are heterogeneously dispersed both on the outer surface and in the interior of the resin beads, and accumulate only when loading the conditioned CHO cell culture supernatant. Insignificant accumulation is seen if the process is run with purified mAb or with the null cell culture supernatant. When bound to the Protein A ligand, the mAb responsible for the observed fouling behavior is shown to associate with BSA and α-lactalbumin. This property is exploited using labeled versions of these lipophilic proteins to assess the effectiveness of improved resin cleaning processes and to elucidate the fouling mechanism. Resin fouling for this mAb appears to be consistent with the occurrence of conformational changes that occur upon binding, which, in turn, facilitate association of lipophilic proteins with the mAb. Upon desorption at low pH, these destabilized mAb complexes are deposited on and within the resin growing with each cycle and eventually leading to significant degradation of process performance.

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.

A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode.

A significant consequence of scaling up production of high titer monoclonal antibody (mAb) processes in existing facilities is the generation of in-process pools that exceed the capacity of storage vessels. A semi-continuous downstream process where columns and filters are linked and operated in tandem would eliminate the need for intermediate holding tanks. This study is a bench-scale demonstration of the feasibility of a tandem process for the purification of mAbs employing an affinity Protein A capture step, followed by a flow-through anion-exchange (AEX) step with the possibility of adding an in-line virus filtration step (VF). All three steps were linked sequentially and operated as one continuous process using an ÄKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX column was operated in a weak partitioning (WP) mode enabled by a precise in-line titration of Protein A effluent. In order to avoid complex control schemes and facilitate validation, quality and robustness were built into the system through selection of buffers based on thermodynamic and empirical models. The tandem system utilized the simplest possible combination of valves, pumps, controls, and automation, so that it could easily be implemented in a clinical or commercial production facility. Linking the purification steps in a tandem process is expected to generate savings in time and production costs and also reduce the size of quality systems due to reduced documentation requirements, microbial sampling, and elimination of hold time validation.

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.

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography.

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product Kp value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425-434, 2017.

Mechanistic failure mode investigation and resolution of parvovirus retentive filters.

Virus retentive filters are a key product safety measure for biopharmaceuticals. A simplistic perception is that they function solely based on a size-based particle removal mechanism of mechanical sieving and retention of particles based on their hydrodynamic size. Recent observations have revealed a more nuanced picture, indicating that changes in viral particle retention can result from process pressure and/or flow interruptions. In this study, a mechanistic investigation was performed to help identify a potential mechanism leading to the reported reduced particle retention in small virus filters. Permeate flow rate or permeate driving force were varied and analyzed for their impact on particle retention in three commercially available small virus retentive filters. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:959-970, 2016.

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode.

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow-rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log10 of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach.

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.

Structure and protein adsorption mechanisms of clean and fouled tentacle-type anion exchangers used in a monoclonal antibody polishing step.

The properties of Fractogel(®) EMD TMAE HiCap (M), a tentacle-type anion exchange resin used for a polishing step in a monoclonal antibody (mAb) purification process, were investigated for both virgin and used samples to determine the influence of process related fouling. Inverse size exclusion chromatography indicated a bimodal distribution of pore sizes consisting mostly of small pores, 4-5 nm in radius and likely associated with the grafted tentacles. Similar results were obtained for resin samples fouled by process use, indicating that the core structure of these particles is unchanged. Transmission electron micrographs showed that the resin backbone matrix has a microgranular structure. However, a dense skin layer, 0.2-0.5 µm thick, was also seen at the exterior surface of the fouled particles. The binding capacity attained for BSA after 90 min of contact was 165 ± 4 mg/mL for both virgin and fouled samples, close to the equilibrium capacity of 178 ± 2 mg/mL attained after 24h. On the other hand, the capacities attained at 90 min for the much larger thyroglobulin were only 90 ± 4 and 25 ± 2 mg/mL, respectively, for virgin and fouled samples. The BSA adsorption kinetics was also slower for the fouled resin, but much larger kinetic differences between virgin and fouled resin were seen for thyroglobulin. Based on the shape of intraparticle protein concentration profiles determined by confocal laser scanning microscopy (CLSM), the protein transport mechanism is consistent with solid diffusion for both virgin and fouled resin samples and proteins. However, transport is hindered by the foulant layer to a much greater extent for thyroglobulin as a result of its larger size. Additional measurements indicated that the foulant layer is consistent with mAb aggregates irreversibly bound at the particle exterior surface.

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.