Simulation of continuous low pH viral inactivation inside a coiled flow inverter

Continuous production of monoclonal antibodies is gaining more and more importance. To ensure continuous flow through the entire process as well as viral safety, continuous viral clearance needs to be investigated as well. This study focuses on low pH viral inactivation inside a coiled flow inverter (CFI). Computational fluid dynamics (CFD) simulation is used to gain further insight into the inactivation process inside the apparatus. The influence of viruses in comparison to different tracer elements on the residence time distribution (RTD) behavior is investigated. Finally, the viral inactivation kinetics are implemented into the CFD simulation and real process conditions are simulated. These are compared to experimental results. To the authors' knowledge, this study represents the first successful simulation of continuous viral inactivation inside a CFI. It allows the detailed analysis of processes inside the apparatus and the prediction of experimental virus study results and will therefore contribute to the effective planning of future validation studies.     

Simulation of pH level distribution inside a coiled flow inverter for continuous low pH viral inactivation

The continuous production of monoclonal antibodies (mAbs) with the help of disposable equipment poses one of the future major changes in the pharmaceutical industry. Consequently, continuous viral clearance needs to be developed as well. The coiled flow inverter (CFI) was successfully implemented in the continuous downstream as a residence time module for low pH viral inactivation. As the elution profile of the upstream continuously operated protein A chromatography results in fluctuating pH values, the pH level distribution inside the CFI is highly relevant. This study presents a detailed investigation of pH level distribution inside the CFI at varying inlet conditions with the help of computational fluid dynamics simulation. The simulation model was validated first with the help of experimental data. Afterwards, the model was used for further investigations. It was determined that with a pH sine curve as input, the duration until steady state at the outlet requires two times the minimum residence time of the apparatus. Moreover, it could be observed that the CFI itself offers a progressive dampening effect on the pH level distribution. Afterwards, different forms of the sine curve representing different operation modes of the continuous protein A chromatograph were tested to evaluate this dampening capability. It became clear that the switch time has the highest influence on the resulting pH of the outlet stream and should be considered for process development. Finally, the radial pH profiles at different positions inside the CFI were determined. This once again revealed the high radial mixing capability of the CFI and its influence on the resulting product stream.     

Truly continuous low pH viral inactivation for biopharmaceutical process integration

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities—which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.     

Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by d -sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields.     

Characterization of operating parameters for XMuLV inactivation by low pH treatment

To ensure the viral safety of protein therapeutics made in mammalian cells, purification processes include dedicated viral clearance steps to remove or inactivate adventitious and endogenous viruses. One such dedicated step is low pH treatment, a robust and effective method commonly used in monoclonal antibody production to inactivate enveloped viruses. To characterize the operating space for low pH viral inactivation, we performed a statistically designed experiment evaluating the effect of pH, temperature, hold duration, acid type, and buffer concentration on inactivation of the retrovirus model, XMuLV. An additional single factor experiment was performed to study the effect of protein concentration. These data were used to generate predictive models of inactivation at each time point studied, which can be used to identify conditions for robust and effective XMuLV inactivation. At pH 3.6, XMuLV inactivation was rapid, robust, and relatively unaffected by the other factors studied, providing support for this as a generic viral inactivation condition for products that can tolerate this low pH. At pH 3.7 and 3.8, other factors besides pH affected XMuLV inactivation. By understanding the impact of each factor on inactivation, the factors can be manipulated within the operating space to ensure effective inactivation while achieving desired product quality goals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:89–97, 2016     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

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.     

Protein aggregation and mitigation strategy in low pH viral inactivation for monoclonal antibody purification

ABSTRACT

Significant amounts of soluble product aggregates were observed during low-pH viral inactivation (VI) scale-up for an IgG4 monoclonal antibody (mAb IgG4-N1), while small-scale experiments in the same condition showed negligible aggregation. Poor mixing and product exposure to low pH were identified as the root cause. To gain a mechanistic understanding of the problem, protein aggregation properties were studied by varying critical parameters including pH, hold time and protein concentration. Comprehensive biophysical characterization of product monomers and aggregates was performed using fluorescence-size-exclusion chromatography, differential scanning fluorimetry, fluorescence spectroscopy, and dynamic light scattering. Results showed IgG4-N1 partially unfolds at about pH 3.3 where the product molecules still exist largely as monomers owing to strong inter-molecular repulsions and favorable colloidal stability. In the subsequent neutralization step, however, the conformationally changed monomers are prone to aggregation due to weaker inter-molecular repulsions following the pH transition from 3.3 to 5.5. Surface charge calculations using homology modeling suggested that intra-molecular repulsions, especially between CH2 domains, may contribute to the IgG4-N1 unfolding at ≤ pH 3.3. Computational fluid dynamics (CFD) modeling was employed to simulate the conditions of pH titration to reduce the risk of aggregate formation. The low-pH zones during acid addition were characterized using CFD modeling and correlated to the condition causing severe product aggregation. The CFD tool integrated with the mAb solution properties was used to optimize the VI operating parameters for successful scale-up demonstration. Our research revealed the governing aggregation mechanism for IgG4-N1 under acidic conditions by linking its molecular properties and various process-related parameters to macroscopic aggregation phenomena. This study also provides useful insights into the cause and mitigation of low-pH-induced IgG4 aggregation in downstream VI operation.     

Leveraging flow mechanics to determine critical process and scaling parameters in a continuous viral inactivation reactor

A continuous viral inactivation (CVI) chamber has been designed to operate with acceptable residence time distribution (RTD) characteristics. However, altering the CVI's geometry and operation to accommodate the scale was not obvious. In this work, we elucidate the influence of Dean vortices and leverage the transition into the weak turbulent regime to establish relationships between input variables and process outputs. This study was targeted to understand and quantify the impact of viscosity, Dean number, internal diameter, and path length on the RTD. When the Dean number exceeds 70, radial mixing generated by the Dean vortices began to consistently alter the axial dispersive effects experienced by the pulse injection. Increasing to a Dean number of >100, the axial dispersive effects were dominated by the Dean vortices which allowed the calculation of the minimum and maximum residence time to be generated. This work provides a method to calculate operational solutions for a tubular incubation reactor in terms of path length, internal diameter, flow rate, and target minimum and maximum residence time specifications that assures both viral residence times while also establishing criteria to maximize product quality during continuous operation.     

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end‐to‐end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three‐column periodic counter‐current chromatography for capture of the product followed by a three‐column con‐current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end‐to‐end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998–1009, 2017     

Design,construction, and optimization of a novel,modular, and scalable incubation chamber for continuous viral inactivation

We designed, built or 3D printed, and screened tubular reactors that minimize axial dispersion to serve as incubation chambers for continuous virus inactivation of biological products. Empirical residence time distribution data were used to derive each tubular design's volume equivalent to a theoretical plate (VETP) values at a various process flow rates. One design, the Jig in a Box (JIB), yielded the lowest VETP, indicating optimal radial mixing and minimal axial dispersion. A minimum residence time (MRT) approach was employed, where the MRT is the minimum time the product spends in the tubular reactor. This incubation time is typically 60 minutes in a batch process. We provide recommendations for combinations of flow rates and device dimensions for operation of the JIB connected in series that will meet a 60‐min MRT. The results show that under a wide range of flow rates and corresponding volumes, it takes 75 ± 3 min for 99% of the product to exit the reactor while meeting the 60‐min MRT criterion and fulfilling the constraint of keeping a differential pressure drop under 5 psi. Under these conditions, the VETP increases slightly from 3 to 5 mL though the number of theoretical plates stays constant at about 1326 ± 88. We also demonstrated that the final design volume was only 6% ± 1% larger than the ideal plug flow volume. Using such a device would enable continuous viral inactivation in a truly continuous process or in the effluent of a batch chromatography column. Viral inactivation studies would be required to validate such a design. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:954–965, 2017     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

A rotating disk electrode for kinetic studies of superoxide dismutases: applicability in a wide pH range and for continuous monitoring of enzyme inactivation

A rotating disk electrode coated with a thin mercury film is described. It is suitable for electrochemical determinations of the catalytic constant of superoxide dismutase in a much wider pH range than that accessible to polarographic methods. In particular this is the only direct method, except for pulse radiolysis, that allows mechanistic studies of the enzyme in the physiological pH range. Furthermore, the continuous response of this electrode allows enzyme activity changes characterized by half-lives in the range from a few to 10(3) s. to be followed.     

Computational prediction of blend time in a large-scale viral inactivation process for monoclonal antibodies biomanufacturing

Viral inactivation (VI) is a process widely used across the pharmaceutical industry to eliminate the cytotoxicity resulting from trace levels of viruses introduced by adventitious agents. This process requires adding Triton X-100, a non-ionic detergent solution, to the protein solution and allowing sufficient time for this agent to inactivate the viruses. Differences in process parameters associated with vessel designs, aeration rate, and many other physical attributes can introduce variability in the process, thus making predicting the required blending time to achieve the desired homogeneity of Triton X-100 more critical and complex. In this study we utilized a CFD model based on the lattice Boltzmann method (LBM) to predict the blend time to homogenize a Triton X-100 solution added during a typical full-scale commercial VI process in a vessel equipped with an HE-3-impeller for different modalities of the Triton X-100 addition (batch vs. continuous). Although direct experimental progress of the blending process was not possible because of GMP restrictions, the degree of homogeneity measured at the end of the process confirmed that Triton X-100 was appropriately dispersed, as required, and as computationally predicted here. The results obtained in this study were used to support actual production at the biomanufacturing site.     

Influence of pH and sodium chloride on kinetics of thermal inactivation of the bacteriocin-like substance P34

Aims: To investigate the kinetics of thermal inactivation of the bacteriocin‐like substance P34 at different pH and sodium chloride concentration. Methods and Results: Samples of bacteriocin were treated at different time–temperature combinations in the range of 0–300 min and 90–120°C and the kinetic parameters for bacteriocin inactivation were calculated. For all treatments, the thermal inactivation reaction fitted adequately to first‐order model. D‐ and k‐values were smaller and higher, respectively, for pH 4·5 than for 6·0 or 7·0, indicating that bacteriocin P34 was less thermostable at lower pH. At 120, 115 and 100°C, the addition of sodium chloride decreased thermal stability. For other temperatures, addition of NaCl increased stability of the peptide. The presence of greater amount of the salt (50 g l^?1) resulted in a higher thermal stability of bacteriocin P34, suggesting that the reduction in water activity of the solution interfered on the stability of the peptide. Conclusions: Based on an isothermal experiment in the temperature range of 90–120°C, and by thermal death time models, bacteriocin P34 is less heat stable at low pH and has increased thermal stability in the presence of NaCl. Addition of NaCl improved the stability of the peptide P34 at high temperatures. Significance and Impact of the Study: Studies on kinetics of thermal inactivation of bacteriocins are essential to allow their proper utilization in the food industry.     

Economic assessment of continuous processing for manufacturing of biotherapeutics

Continuous processing offers a promising approach to revolutionize biotherapeutics manufacturing as reflected in recent years. The current study offers a comparative economic assessment of batch and continuous processing for the production of biotherapeutic products. Granulocyte-colony stimulating factor (GCSF), a protein expressed in E. coli, and an IgG1 monoclonal antibody, were chosen as representatives of microbial and mammalian derived products for this assessment. Economic indicators—cost of goods (COGs), net present value (NPV), and payback time have been estimated for the assessment. For the case of GCSF, conversion from batch to integrated continuous manufacturing induced a $COGs/g reduction of 83% and 73% at clinical and commercial scales, respectively. For the case of mAb therapeutic, a 68% and 35% reduction in $COGs/g on translation from batch to continuous process was projected for clinical and commercial scales, respectively. Upstream mAb titer was also found to have a significant impact on the process economics. With increasing mAb titer, the $COG/g decreases in both operating modes. With titer increasing from 2 to 8 g/L, the $COG/g of batch process was reduced by 53%, and that of the continuous process was reduced by 63%. Cost savings in both the cases were attributed to increased productivity, efficient equipment and facility utilization, smaller facility footprint, and reduction in utilization of consumables like resin media and buffers actualized by the continuous processing platform. The current study quantifies the economic benefits associated with continuous processing and highlights its potential in reducing the manufacturing cost of biotherapeutics.     

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

A novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution

Charge heterogeneity of monoclonal antibodies is considered a critical quality attribute and hence needs to be monitored and controlled by the manufacturer. Typically, this is accomplished via separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality upon slightest changes in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. In this study, we propose a novel approach for separation of charge variants during continuous CEX chromatography by utilizing a combination of displacement mode chromatography and salt-based step elution. It has been demonstrated that while the displacement mode of chromatography enables control of acidic variants ≤26% in the CEX eluate, salt-based step gradient elution manages basic charge variant ≤25% in the CEX eluate. The proposed approach has been successfully demonstrated using feed materials with varying compositions. On comparing the designed strategy with 2-column concurrent (CC) chromatography, the resin specific productivity increased by 95% and resin utilization increased by 183% with recovery of main species >99%. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was examined in our in-house continuous mAb platform.