Tracking raw material flow through a continuous direct compression line. Part II of II: Predicting dynamic changes in quality attributes of tablets due to disturbances in raw material properties using an independent residence time distribution model.

Continuous manufacturing of pharmaceuticals promises many advantages regarding economics and quality. However, tracing deviating material in such processes is much more challenging than in batch processes due to axial back-mixing. The literature has proven the traceability of disturbances in the active pharmaceutical ingredient (API) by residence time distribution (RTD) models. Nevertheless, pharmaceutical quality attributes (QAs) and disturbances are not limited to the API content. The present study investigates different disturbances affecting the particle size in a direct compression by recording various QAs. The application of a tracer-based model demonstrates the generalizability of RTD models regarding tracing the propagation of nonconforming material. The model applied may predict the appearance of deviating material in the tablets accurately up to 60  s. However, nonlinear and dynamic effects complicate predicting values of the QAs. Due to dynamic effects, tablet masses deviate up to 2.5 percentage points from values measured in steady-state, at an acceptable total deviation of 7.5 %. Consequently, if other QAs are critical, the transfer of control concepts developed for the API content will be challenging due to such effects different from API-based disturbances. Additionally, higher tolerances for errors will be required considering those deviations.

Tracking raw material flow through a continuous direct compression line Part I of II: Residence time distribution modeling and sensitivity analysis enabling increased process yield.

Continuous manufacturing (CM) offers advantages in quality and space-time yield compared to common batch manufacturing. However, higher yield losses due to the start-up procedure make a broad application uneconomical. This work discusses the possibility of reducing yield losses by adjusting the degree of back-mixing. Back-mixing of nonconforming material from disturbances or start-up will result in the contamination of subsequent material. Therefore, higher degrees of back-mixing cause the discharge of additional material. Choosing an advantageous setting of operational parameters may be a simple way to change the degree of back-mixing. Based on direct compression, this work demonstrates the identification of promising parameters. Therefore, step-change experiments using color-marked material in the feeder, blender, and tablet press quantify the impact of three operational parameters per device. Models for the devices and the entire process result from those measurements. Subsequently, a global variance-based sensitivity analysis identifies the most influential parameters. As a result, adjusting the minimal filling level of the feeder and the rotational feed frame speed of the tablet press reduces back-mixing by more than 30 %. At high costs of the raw materials, the resulting savings can significantly improve the economic performance of CM compared to batch manufacturing.

Challenges in tracing material flow passing a loss-in-weight feeder in continuous manufacturing processes.

Loss-in-weight feeders are an integral part of most continuous manufacturing processes, ensuring a constant mass flow. The feeders cause a significant degree of back-mixing in such lines. Understanding back-mixing is essential for the treatment of disturbances. However, feeders refilled semi-continuously contradict the common theory assuming steady-state. This study aims at understanding dynamic back-mixing and related phenomena. Low filling levels of a feeder are investigated using a fluorescent tracer. These investigations prove an impact of the addition of material probably caused by a non-uniform draw-in of the screws and dead material in the hopper. In turn, the dead material accounts for up to 50 % of the material in the hopper. Possible evidence of dead zones at higher filling levels and in feeders from literature are discussed additionally. Steady-state models from literature are extended to represent the observations and back-mixing at all filling levels. This extension reduces the root-mean-squared deviation of the model from the experimental data by 41%. The model predicts different responses to similar disturbances depending on the filling. This state-dependent back-mixing and the observed dead zones are challenging for diverting non-conforming material and material traceability. Therefore, these phenomena should be considered in selecting and operating feeders.

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies.

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.

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.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha.

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Display of malaria transmission-blocking antigens on chimeric duck hepatitis B virus-derived virus-like particles produced in Hansenula polymorpha.

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS: We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS: In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION: The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

Recovery of Natural α-Ionone from Fermentation Broth.

Recently, the market value of aromas has constantly been rising. Because the supply from natural feedstock is limited, the biotechnological production has received more interest. Thus far, only a few attempts have been made to produce α-ionone, a valued essential aroma of raspberry, biotechnologically. This study reports a production process for enantiopure (R)-α-ionone from lab scale (2-150 L) with typical titer of 285 mg/L broth to industrial scale (up to 10 000 L) with a titer up to 400 mg/L broth, focusing on the development of a downstream process with a maximized yield at minimized effort. The developed recovery consists of solid-liquid extraction from the biomass at φ = 0.4 g of n-hexane/g of biomass for 90 min at ambient temperature and adsorption from the aqueous supernatant at Φ = 0.5 g of Diaion HP-20/mg of α-ionone, followed by desorption at Ψ = 30 g of n-hexane/g of Diaion HP-20. Altogether, natural α-ionone could be gained in substantial quantity and purity of >95%.

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.

Establishment of a yeast-based VLP platform for antigen presentation.

BACKGROUND: Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. RESULTS: In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. CONCLUSIONS: The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

Efficient conversion of pretreated brewer's spent grain and wheat bran by submerged cultivation of Hericium erinaceus.

Brewer's spent grain (BSG) and wheat bran (WB) are industrial byproducts that accumulate in millions of tons per year and are typically applied as animal feed. Since both byproducts show a great potential as substrates for fermentation, the approach developed in this study consists of utilizing these lignocellulosic byproducts for biomass production of the medicinal fungus Hericium erinaceus through submerged cultivation. To increase the biological efficiency of the bioconversion, acidic pretreatment was applied yielding a bioconversion of 38.6% for pretreated BSG and 34.8% for pretreated WB. This study shows that the complete degradation of (hemi)cellulose into monosaccharides was not required for an efficient bioconversion. The produced fungal biomass was applied in a second fermentation step to induce the secondary metabolite erinacine C production. Thus, biomass was produced as a functional food ingredient with erinacine C contents of 174.8mg/g for BSG and 99.3mg/g for WB based bioconversions.

Erinacine C: A novel approach to produce the secondary metabolite by submerged cultivation of Hericium erinaceus.

Erinacine C is a cyathane scaffold-based secondary metabolite, which is naturally produced by the filamentous fungus Hericium erinaceus and has a high potential to treat nervous diseases such as Alzheimer's disease. The investigated approach consists of combining an optimised precultivation of H. erinaceus with an enhanced erinacine C production by developing a suitable main cultivation medium enabling the utilisation of high biomass contents. The final erinacine C production medium is buffered by 100 mM HEPES to ensure a stable pH value of 7.5 during main cultivation at inoculation ratios of up to 5:10 (v/v). The medium components, such as 5.0 g L(-1) oatmeal, 1.5 g L(-1) calcium carbonate, and 0.5 g L(-1) Edamin(®) K are crucial for an increased erinacine C production. Besides, different carbon to nitrogen ratios of 25, 64, and 103 do not affect the erinacine C synthesis. The investigated approach enables the production of 2.73 g erinacine C per litre main cultivation broth, which is tenfold higher than published data. In addition, erinacine C biosynthesis is determined to occur mainly in the first six days of main cultivation.

Developing the biofacility of the future based on continuous processing and single-use technology.

To maintain or strengthen their market position, biopharmaceutical producers have to adapt their production facilities to a drastically changed market environment. Contrary to currently used large scale batch-wise operated production facilities, where stainless steel equipment is widely applied, small scale and flexible production processes are desired. Consequently, the concept of the "biofacility of the future" has been developed, which combines the attributes fast, flexible, small, inexpensive and sustainable. Four design principles build the facility's basis and are presented within this work: continuous processing, 100% single-use equipment, closed processing and adopting the ballroom concept. However, no publication presents a completely continuously operated platform process for the production of monoclonal antibodies up to now. Therefore, this work establishes the proof of concept regarding continuous antibody manufacturing. A pilot plant for the production of monoclonal antibodies has been built 100% in single-use equipment. It was operated fully continuous and automated in the upstream and the downstream part. The concepts that allow continuously operating the pilot plant are presented within this work, i.e., continuously operated filtration, continuously operated viral inactivation, continuously operated chromatography and a continuously operated formulation. Analytics showed that the produced product was within specification limits of industrial bulk drug substances.

Heterologous fermentation of a diterpene from Alternaria brassisicola.

A variety of different applications render terpenes and terpenoids attractive research targets. A promising but so far insufficiently explored family of terpenoids are the fusicoccanes that comprise a characteristic 5-8-5 fused tricyclic ring system. Besides herbicidal effects, these compounds also show apoptotic and anti-tumour effects on mammalian cells. The access to fusicoccanes from natural sources is scarce. Recently, we introduced a metabolically engineered Saccharomyces cerevisiae strain to enable the heterologous fermentation of the shared fusicoccane-diterpenoid precursor, fusicocca-2,10(14)-diene. Here, we show experiments towards the identification of bottlenecks in this process. The suppression of biosynthetic by-products via medium optimisation was found to be an important aspect. In addition, the fermentation process seems to be improved under oxygen limitation conditions. Under fed-batch conditions, the fermentation yield was reproducibly increased to approximately 20 mg/L. Furthermore, the impact of the properties of the terpene synthase on the fermentation yield is discussed, and the preliminary studies on the engineering of this key enzyme are presented.

Molecular interactions of alcohols with zeolite BEA and MOR frameworks.

Zeolites can adsorb small organic molecules such as alcohols from a fermentation broth. Also in the zeolite-catalyzed conversion of alcohols to biofuels, biochemicals, or gasoline, adsorption is the first step. Several studies have investigated the adsorption of alcohols in different zeolites experimentally, but computational investigations in this field have mostly been restricted to zeolite MFI. In this study, the adsorption of C1-C4 alcohols in BEA and MOR was investigated using density functional theory (DFT). Calculated adsorption geometries and the corresponding energies of the designed cluster models were comparable to periodic calculations, and the adsorption energies were in the same range as the corresponding computational and experimental values reported in the literature for zeolite MFI. Thus, BEA and MOR may be good adsorption materials for alcohols in the field of downstream processing and catalysis. Aside from the DFT calculations, adsorption isotherms were determined experimentally in this study from aqueous solutions. For BEA, the adsorption of significant amounts of alcohol from aqueous solution was observed experimentally. In contrast, MOR was loaded with only a very small amount of alcohol. Although differences were found between the affinities obtained from gas-phase DFT calculations and those observed experimentally in aqueous solution, the computational data presented here represent molecular level information on the geometries and energies of C1-C4 alcohols adsorbed in zeolites BEA and MOR. This knowledge should prove very useful in the design of zeolite materials intended for use in adsorption and catalytic processes, as it allows adsorption behavior to be predicted via judiciously designed computational models.

Investigation of structural changes of ß-casein and lysozyme at the gas-liquid interface during foam fractionation.

Purification and separation of proteins play a major role in biotechnology. Nowadays, alternatives to multistep operations suffering from low product yields and high costs are investigated closely amidst which one of the promising options is foam fractionation. The molecular behavior at the gas-liquid interface plays an important role in the formation and stabilization of enriched foam. This study for the first time correlates the physico-chemical parameters to the molecular structure in view of protein enrichment during foam fractionation of the two relatively different proteins lysozyme and ß-casein employing biophysical techniques such as circular dichroism (CD) spectroscopy and infrared reflection absorption spectroscopy (IRRAS). In case of lysozyme, high enrichment was achieved at pH

Fugitive emissions from liquid-charged flange joints: a comparison of laboratory and field data.

Starting from Hagen-Poiseuille's law, we analyzed the parameters influencing fugitive emission rates. It is shown that liquid and gas emissions follow basically different mass transport mechanisms so that mathematical models available for gases are not suitable to calculate liquid emissions. The emission behavior of liquid-charged flange joints is characterized, and a model conception for the observed phenomena is presented explaining the unexpectedly high emissions of liquid-charged flange joints. Against the background of field data, the influence of maintenance on fugitive emmissions is demonstrated, and exchanging old gaskets with high-grade gaskets as a means of emission reduction in old plants is critically scrutinized. A general maintenance plan is proposed as the most effective method for emission reductions.