Novel spiking methods developed for anion exchange chromatography operating in a continuous process

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a nonhomogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in-line spiking. This is challenging to be implemented at the contract research organization performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow-through anion exchange chromatography step in a monoclonal antibody (mAb) manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of anion exchange chromatography unit operating in continuous processing.     

Virus clearance validation across continuous capture chromatography

Multicolumn capture chromatography is gaining increased attention lately due to the significant economic and process advantages it offers compared with traditional batch mode chromatography. However, for wide adoption of this technology in clinical and commercial space, it requires scalable models for executing viral validation studies. In this study, viral validation studies were conducted under cGLP guidelines to assess retro- (X-MuLV) and parvo-virus (MVM) clearance across twin-column continuous capture chromatography (CaptureSMB). A surrogate model was also developed using standard batch mode chromatography based on flow path modifications to mimic the loading strategy used in CaptureSMB. The results show that a steady state was achieved by the second cycle for both antibody binding and virus clearance and that the surrogate model using batch mode chromatography equipment provided impurity clearance that was comparable to that obtained during cyclical operation of CaptureSMB. Further, the log reduction values (LRVs) achieved during CaptureSMB were also comparable to the LRVs obtained using standard batch capture chromatography. This was expected since the mode of virus separation during protein A chromatography is primarily based on removal during the flow through and wash steps. Finally, this study also presents assessments on the resin cleaning strategy during continuous chromatography and how the duration of clean-in-place solution exposure impacts virus carryover.     

A continuous multicolumn countercurrent solvent gradient purification (MCSGP) process

Biomolecules are often purified via solvent gradient batch chromatography. Typically suitable smooth linear solvent gradients are applied to obtain the separation between the desired component and hundreds of impurities. The desired product is usually intermediate between weakly and strongly adsorbing impurities, and therefore a central cut is required to get the desired pure product. The stationary phases used for preparative and industrial separations have a low efficiency due to strong axial dispersion and strong mass transfer resistances. Therefore a satisfactory purification often cannot be achieved in a single chromatographic step. For large scale productions and for very valuable molecules, countercurrent operation such as the well known SMB process, is needed in order to increase separation efficiency, yield and productivity. In this work a novel multicolumn solvent gradient purification process (MCSGP-process) is introduced, which combines two chromatographic separation techniques, which are solvent gradient batch and continuous countercurrent SMB. The process consists of several chromatographic columns, which are switched in position opposite to the flow direction. Most of the columns are equipped with a gradient pump to adjust the modifier concentration at the column inlet. Some columns are interconnected, so that non pure product streams are internally, countercurrently recycled. Other columns are short circuited and operate in batch mode. As a working example the purification of an industrial stream containing 46% of the hormone Calcitonin is considered. It is found that for the required purity the MCSGP unit achieves a yield close to 100% compared to a maximum value of a single column batch chromatography of 66%.     

Virus study for continuous low pH viral inactivation inside a coiled flow inverter

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation.     

Process for Continuous Fab Production by Digestion of IgG

Intensified processing and end‐to‐end integrated continuous manufacturing are increasingly being considered in bioprocessing as an alternative to the current batch‐based technologies. Similar approaches can also be used at later stages of the production chain, such as in the post‐translational modifications that are often considered for therapeutic proteins. In this work, a process to intensify the enzymatic digestion of immunoglobulin G (IgG) and the purification of the resulting Fab fragment is developed. The process consists of the integration of a continuous packed‐bed reactor into a multicolumn chromatographic process. The integration is realized through the development of a novel multicolumn countercurrent solvent gradient purification (MCSGP) process, which, by adding a third column to the classical two‐column MCSGP process, allows for continuous loading and then straight‐through processing of the mixture leaving the reactor.     

Adapting virus filtration to enable intensified and continuous monoclonal antibody processing

Ongoing efforts in the biopharmaceutical industry to enhance productivity and reduce manufacturing costs include development of intensified, linked, and/or continuous processes. One approach to improve productivity and process economics of the polishing step (i.e., anion exchange chromatography) is to preconcentrate the product intermediate using a single-pass tangential flow filtration step before loading on the resin. This intensification of the polishing step consequently leads to changes in product intermediate concentration for subsequent virus filtration operations, potentially impacting filter performance and methods for evaluating viral clearance. The filtrate flux performance of a virus filtration operation was evaluated with monoclonal antibody (mAb) solutions of varying concentrations. These data were used to evaluate the effect on filter sizing for a hypothetical mAb perfusion process. The optimum mAb concentration to minimize the area of the virus filter was a function of the filtration step duration and reflected the competing effects of increasing concentration and decreasing volumetric flux on the membrane productivity. mAb solutions at high and low concentrations were used to evaluate viral clearance with extended filtration times (e.g., 24–72 h) simulating continuous processing conditions. Modifications to more traditional filtration viral clearance study methods were required to avoid experimental artifacts associated with the extended filtration time. No virus passage through the filter was observed under these conditions, similar to previous results for batch processes. These data demonstrate the feasibility of obtaining effective virus removal even when mAb concentration and filtrations times are increased by up to an order of magnitude from current common practices.     

Continuous precipitation of process related impurities from clarified cell culture supernatant using a novel coiled flow inversion reactor (CFIR)

Coiled Flow Inverter Reactor (CFIR) has recently been explored for facilitating continuous operation of several unit operations involved in downstream processing of biopharmaceuticals such as viral inactivation and protein refolding. The application of CFIR for continuous precipitation of clarified cell culture supernatant has been explored. The pH based precipitation is optimized in the batch mode and then in the continuous mode in CFIR using a design of experiments (DOE) study. Improved clearance of host cell DNA (52× vs. 39× in batch), improved clearance of host cell proteins (HCP) (7× vs. 6× in batch) and comparable recovery (90 vs. 91.5 % in batch) are observed along with six times higher productivity. To further demonstrate wider applicability of CFIR in performing continuous precipitation, two more case studies involving use of two different precipitation protocols (CaCl₂ based and caprylic acid based) are also performed. In both cases, clearance of host cell DNA, HCP, and product recovery are found to be comparable or better in CFIR than in batch operations. Moreover, increase in productivity of 16 times (CaCl₂ based) and eight times (caprylic acid based) is obtained for the two precipitation protocols, respectively. The data clearly demonstrate that CFIR can be seamlessly integrated into a continuous bioprocess train for performing continuous precipitation of clarified cell culture supernatant. To our knowledge this is the first report of such use.     

Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode

The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.     

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log₁₀ reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65–3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.     

Analysis of viral clearance unit operations for monoclonal antibodies

Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody‐related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late‐phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro‐ and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log₁₀ were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses. Biotechnol. Bioeng. 2010;106: 238–246. Published 2010 Wiley Periodicals, Inc.     

Integrated continuous production of recombinant therapeutic proteins

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost‐effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high‐density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four‐column periodic counter‐current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high‐density perfusion CHO cell cultures were operated at a quasi‐steady state of 50–60 × 10⁶ cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed‐batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time‐based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch‐column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non‐value‐added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. Biotechnol. Bioeng. 2012; 109: 3018–3029. © 2012 Wiley Periodicals, Inc.     

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis,Characterization, and Purification

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom‐made drugs (also called N‐to‐1) able to act on the gene expression at pre‐translational level. With recent approvals of ON‐based therapeutics by the Food and Drug Administration (FDA), a growing demand for high‐quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market‐size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid‐phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single‐column operations. This consideration is supported by a recent cutting‐edge application of continuous chromatography for the ON purification.     

Mechanistic insights into viral clearance during the chromatography steps in antibody processes by using virus surrogates

Viral safety is required for biological products to treat human diseases, and the burden of inactivation and or virus removal lies on the downstream purification process. Minute virus of mice (MVM) is a nonenveloped parvovirus commonly used as the worst-case model virus in validation studies because of its small size and high chemical stability. In this study, we investigated the use of MVM-mock virus particle (MVP) and bacteriophage ΦX174 as surrogates for MVM to mimic viral clearance studies, with a focus on chromatography operations. Based on structural models and comparison of log reduction value among MVM, MVP, and ΦX174, it was demonstrated that MVP can be used as a noninfectious surrogate to assess viral clearance during process development in multiple chromatography systems in a biosafety level one (BSL-1) laboratory. Protein A (ProA) chromatography was investigated to strategically assess the impact of the resin, impurities, and the monoclonal antibody product on virus removal.     

Gamma irradiating chromatography columns enables bioburden-free integrated continuous biomanufacturing

An important consideration for integrated continuous biomanufacturing is that the downstream chromatography steps integrated with the bioreactor should maintain a low bioburden state throughout the entire duration of the operation. One potential strategy to achieve this is to start bioburden-free and functionally close the chromatography system. While chromatography skids themselves can be rendered bioburden-free, limitations exist in applying these methods to chromatography columns. The small column sizes used in continuous multicolumn chromatography enable gamma irradiation of disposable columns to render them bioburden-free. However, this approach has not been widely implemented, likely because gamma irradiation can negatively impact resin performance. Here, several protective mobile-phase modifiers were screened and shown to help chromatography resins retain naïve-like performance. Gamma irradiated columns were then integrated into perfusion bioreactors for continuous capture. Successful integrated continuous capture downstream of perfusion bioreactors for greater than 40 days using protein A, custom affinity, and non-affinity capture resins for multiple biologic modalities is demonstrated in development and commercial settings. No indications of time-based performance decline or bioburden growth have been observed. This strategy enables bioburden-free integrated continuous biomanufacturing operations and could allow full process closure and decreased environmental control requirements for facilities; thus, permitting simultaneous multi-product operations in a ballroom arrangement.     

Column flow reactor using acetohydroxyacid synthase I from Escherichia coli as catalyst in continuous synthesis of R-phenylacetyl carbinol

We tested the possibility of utilizing acetohydroxyacid synthase I (AHAS I) from Escherichia coli in a continuous flow reactor for production of R-phenylacetyl carbinol (R-PAC). We constructed a fusion of the large, catalytic subunit of AHAS I with a cellulose binding domain (CBD). This allowed purification of the enzyme and its immobilization on cellulose in a single step. After immobilization, AHAS I is fully active and can be used as a catalyst in an R-PAC production unit, operating either in batch or continuous mode. We propose a simplified mechanistic model that can predict the product output of the AHAS I-catalyzed reaction. This model should be useful for optimization and scaling up of a R-PAC production unit, as demonstrated by a column flow reactor.     

Optimum operating mode for a class of fermentation

This paper is concerned with optimization of the operating mode of a fermentor. Combining the various modes of operation—batch, semibatch, and continuous—the operating pattern which maximizes the desired metabolic product in a single fermentor is determined by using Kelley's transformation method with Pontryagin's maximum principle. Kelley's transformation method is a device which avoids the singular situation which occurs when the usual procedure of selecting the optimal control function by the maximum principle breaks down. This is the case in the problem considered in this paper. For lysine fermentation, the best operating mode depends on the fermentor capacity and operating time. The results of this study are summarized thus: (i) when the operating time is “long enough,” optimal conditions require that continuous operation follows either semibatch and/or batch operation, and (ii) when the fermentor capacity becomes “large enough,” semibatch operation becomes important.     

Design and operation of a continuous integrated monoclonal antibody production process

The realization of an end‐to‐end integrated continuous lab‐scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter‐based cell‐retention, a continuous two column capture process, a virus inactivation step, a semi‐continuous polishing step (twin‐column MCSGP), and a batch‐wise flow‐through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity‐yield trade‐off of classical batch‐wise bind‐elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight‐through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end‐to‐end integration. The steady‐state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303–1313, 2017     

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. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750–757, 2015     

Modeling the Residence Time Distribution of Integrated Continuous Bioprocesses

In response to the biopharmaceutical industry advancing from traditional batch operation to continuous operation, the Food and Drug Administration (FDA) has published a draft for continuous integrated biomanufacturing. This draft outlines the most important rules for establishing continuous integration. One of these rules is a thorough understanding of mass flows in the process. A computer simulation framework is developed for modeling the residence time distribution (RTD) of integrated continuous downstream processes based on a unit‐by‐unit modeling approach in which unit operations are simulated one‐by‐one across the entire processing time, and then combined into an integrated RTD model. The framework allows for easy addition or replacement of new unit operations, as well as quick adjustment of process parameters during evaluation of the RTD model. With this RTD model, the start‐up phase to reach steady state can be accelerated, the effects of process disturbances at any stage of the process can be calculated, and virtual tracking of a section of the inlet material throughout the process is possible. A hypothetical biomanufacturing process for an antibody was chosen for showcasing the RTD modeling approach.