Continuous direct compression of a commercially batch-manufactured tablet formulation with two different processing lines.

The transfer from batch-based to continuous tablet manufacturing increases the quality and efficiency of processes. Nonetheless, as in the development of a batch process, the continuous process design requires optimization studies to ensure a robust process. In this study, processing of a commercially batch-manufactured tablet product was tested with two continuous direct compression lines while keeping the original formulation composition and tablet quality requirements. Tableting runs were conducted with different values of process parameters. Changes in parameter settings were found to cause differences in tablet properties. Most of these quality properties could be controlled and maintained within the set limits effortlessly already at this stage of studies. However, the API content and content uniformity seemed to require more investigation. The observed content uniformity challenges were traced to individual tablets with a high amount of API. This was suspected to be caused by API micro-agglomerates since tablet weight variability did not explain the issue. This could be solved by adding a mill between two blenders in the process line. Overall, this case study produced promising results with both tested manufacturing lines since many tablet properties complied with the test result limits without optimization of process parameter settings.

Challenges encountered in the transfer of atorvastatin tablet manufacturing - commercial batch-based production as a basis for small-scale continuous tablet manufacturing tests.

As is the case with batch-based tableting processes, continuous tablet manufacturing can be conducted by direct compression or with a granulation step such as dry or wet granulation included in the production procedure. In this work, continuous manufacturing tests were performed with a commercial tablet formulation, while maintaining its original material composition. Challenges were encountered with the feeding performance of the API during initial tests which required designing different powder pre-blend compositions. After the pre-blend optimization phase, granules were prepared with a roller compactor. Tableting was conducted with the granules and an additional brief continuous direct compression run was completed with some ungranulated mixture. The tablets were assessed with off-line tests, applying the quality requirements demanded for the batch-manufactured product. Chemical maps were obtained by Raman mapping and elemental maps by scanning electron microscopy with energy-dispersive X-ray spectroscopy. Large variations in both tablet weights and breaking forces were observed in all tested samples, resulting in significant quality complications. It was suspected that the API tended to adhere to the process equipment, accounting for the low API content in the powder mixture and tablets. These results suggest that this API or the tablet composition was unsuitable for manufacturing in a continuous line; further testing could be continued with different materials and changes in the process.

Physicochemical stability of high-concentration cefuroxime aqueous injection reconstituted by a centralised intravenous additive service.

OBJECTIVES: Hospital pharmacies provide centralised intravenous additive services (CIVAS), such as antibiotic reconstitution. The aim of this study was to demonstrate the physicochemical stability of high-concentration cefuroxime sodium in aqueous injections, which is mandatory for the centralised preparation of products with automation. METHODS: The physicochemical stability of three high-concentration injections (1.5 g of cefuroxime sodium in 15 mL, 16 mL and 18 mL of water for injection (WFI)) were studied in two primary packing materials (glass vials and polypropylene syringes). The samples were reconstituted with automation in three mid-sized hospital pharmacies in a good manufacturing practice (GMP) grade A/B cleanroom. During the study, the samples were stored in refrigerated conditions (4°C) and 1.5 g/15 mL solution in ambient temperature (22°C). Cefuroxime and descarbamoyl cefuroxime were analysed by high-performance liquid chromatography with UV detection. In addition, the appearance, pH and uniformity of dosage units were investigated. RESULTS: The freshly prepared cefuroxime injections fulfilled the criteria of content uniformity (acceptance value (AV) <15). A significant decrease in concentration of cefuroxime and increase in content of descarbamoyl cefuroxime was observed in all injections. Cefuroxime aqueous injections were physiochemically stable for up to 14 days under refrigeration storage. The relative content of descarbamoyl cefuroxime remained under 3% at 4°C. The solution of 1.5 g/15 mL was stable for only 20 hours in formulations stored for the first 14 days at 4°C and then transferred to 22°C. The colour of the solution changed from light yellow to a darker yellow, and the pH value of the solutions increased during storage. Neither primary packing materials, commercial source of cefuroxime sodium nor exposure to light had any significant effect on the stability of formulations. CONCLUSIONS: Although limited, we found the shelf life of high-concentration cefuroxime injections in refrigerated conditions sufficient for centralised antibiotic preparation in hospital pharmacy with automation. The limited shelf life of high-concentration cefuroxime injections must be considered when using these formulations.

Flowsheet modelling of a powder continuous feeder-mixer system.

In this study, an integrated flowsheet model of the continuous feeder-mixer system was calibrated, simulated and compared against experimental data. The feeding process was first investigated using two major components (ibuprofen and microcrystalline cellulose (MCC)), in a formulation comprised of: 30 wt% of ibuprofen, 67.5 wt% MCC, 2 wt% of sodium starch glycolate and 0.5 wt% of magnesium stearate. The impact of a refill on feeder performance was experimentally evaluated for different operating conditions. Results showed that it had no influence on feeder performance. While simulations with the feeder model fairly reproduced the material behaviour observed in the feeder, unintended disturbances were underpredicted due to the model's low complexity. Experimentally, mixer's efficiency was assessed based on ibuprofen residence time distribution. Mean residence time pointed to a higher mixer's efficiency at lower flow rates. Blend homogeneity results showed that for the entire set of experiments, ibuprofen RSD < 5%, irrespective of process variables. A feeder-mixer flowsheet model was calibrated, after regressing the axial model coefficients. The regression curves exhibited a R2 above 0.96, whereas the RMSE varied from 1.58x10-4 to 1.06x10-3 s-1 across all fitted curves. Simulations confirmed that flowsheet model captured the powder dynamics inside the mixer and qualitatively predicted the mixer's filtering ability against feeding composition fluctuations, as well as ibuprofen RSD in blend, in line with real experiments.

Parameter optimization in a continuous direct compression process of commercially batch-produced bisoprolol tablets.

Continuous tablet manufacturing is a competitive option to replace the traditional batch manufacturing approach. The aim of this study was to evaluate technology transfer from batch-based direct compression of a commercial tablet formulation to continuous direct compression without changes to the composition of the formulation. Some powder studies were conducted with the raw materials and multi-tip punches were utilized in the tableting studies. To lower the high level of tablet weight variability that was evident during preliminary tests, a process parameter optimization was performed using an experimental design with different rpm values of force feeder and mixer impeller. By selecting the most appropriate settings of these parameters for the studied product, the weights of the tablets could be controlled adequately to meet the specification criteria. The functionality of the best-performing parameter settings was investigated with a three-hour-long tableting run. The tablets were evaluated with the same quality criteria as the commercial batch-produced tablets, and they passed all the tests performed in this study. Despite the challenging material properties according to the flowability tests, production of tablets with the desired quality was achieved using the original composition with continuous direct compression.

Detecting different amorphous - Amorphous phase separation patterns in co-amorphous mixtures with high resolution imaging FTIR spectroscopy.

Many active pharmaceutical ingredients (API) in development suffer from low aqueous solubilities. Instead of the crystal form, the amorphous state can be used to improve the API's apparent solubility. However, the amorphous state has a higher Gibb's free energy and is inherently unstable and tends to transform back to the more stable crystal form. In co-amorphous mixtures, phase separation needs to occur before there can be crystallization. The aim of this study was to devise a method to study amorphous-amorphous phase separation with high resolution imaging Fourier transform infrared (FTIR) spectroscopy with seven 1:1 M ratio API-API binary mixtures being examined. The binary mixtures were amorphized by melt-quenching and stored above their glass transition temperature (Tg) to monitor their phase separation. Thermodynamic properties (crystallization tendency, melting point (Tm) and Tg) of these mixtures were measured with differential scanning calorimetry (DSC) to verify the amorphization method and to assess the optimal storage temperature. The phase separation was examined with FTIR imaging in the transmission mode. Furthermore, measurements with two pure APIs were performed to ensure that the alterations occurring in the spectra were caused by phase separation not storage stress. In addition, the reproducibility of the imaging FTIR spectrometer was verified. The spectra were analyzed with principal component analysis (PCA) and a characteristic peak comparison method. Scatter-plots were produced from the amount of phase separated pixels in the measurement area as a way of visualizing the progress of phase separation. The results indicated that imaging with FTIR spectroscopy can produce reproducible results and the progress of phase separation can be detected as either a sigmoidal or as a start-to-finish linear pattern depending on the substances.

Near-infrared analysis of nanofibrillated cellulose aerogel manufacturing.

Biomaterial aerogel fabrication by freeze-drying must be further improved to reduce the costs of lengthy freeze-drying cycles and to avoid the formation of spongy cryogels and collapse of the aerogel structures. Residual water content is a critical quality attribute of the freeze-dried product, which can be monitored in-line with near-infrared (NIR) spectroscopy. Predictive models of NIR have not been previously applied for biomaterials and the models were mostly focused on the prediction of only one formulation at a time. We recorded NIR spectra of different nanofibrillated cellulose (NFC) hydrogel formulations during the secondary drying and set up a partial least square regression model to predict their residual water contents. The model can be generalized to measure residual water of formulations with different NFC concentrations and the excipients, and the NFC fiber concentrations and excipients can be separated with the principal component analysis. Our results provide valuable information about the freeze-drying of biomaterials and aerogel fabrication, and how NIR spectroscopy can be utilized in the optimization of residual water content.

Preservation of biomaterials and cells by freeze-drying: Change of paradigm.

Freeze-drying is the most widespread method to preserve protein drugs and vaccines in a dry form facilitating their storage and transportation without the laborious and expensive cold chain. Extending this method for the preservation of natural biomaterials and cells in a dry form would provide similar benefits, but most results in the domain are still below expectations. In this review, rather than consider freeze-drying as a traditional black box we "break it" through a detailed process thinking approach. We discuss freeze-drying from process thinking aspects, introduce the chemical, physical, and mechanical environments important in this process, and present advanced biophotonic process analytical technology. In the end, we review the state of the art in the freeze-drying of the biomaterials, extracellular vesicles, and cells. We suggest that the rational design of the experiment and implementation of advanced biophotonic tools are required to successfully preserve the natural biomaterials and cells by freeze-drying. We discuss this change of paradigm with existing literature and elaborate on our perspective based on our new unpublished results.

Production and stability of amorphous solid dispersions produced by a Freeze-drying method from DMSO.

Freeze drying is known to be able to produce an amorphous product, but this approach has been mostly used with water-based media. With APIs which are virtually water insoluble, a more appropriate freeze-drying medium would be an organic solvent. Little is known about this approach in terms of forming a stable freeze-dried amorphous product stabilized by small molecule excipient out of organic solvents. In the present study, freeze-drying of APIs from DMSO solutions was used to produce stable solid dispersions from binary mixtures of APIs containing at least one poorly water soluble or practically water-insoluble API. The developed freeze-drying method produced amorphous binary solid dispersions which remained amorphous for at least two days while the 13 best binary dispersions remained stable at room temperature for the entire study period of 127 days. Average residual DMSO levels in dried dispersions were 3.5% ± 1.6%. The developed method proved feasible in producing relatively stable amorphous solid dispersions from practically water insoluble drug compounds which could subsequently be used in further research purposes.

Biopharmaceutics of Topical Ophthalmic Suspensions: Importance of Viscosity and Particle Size in Ocular Absorption of Indomethacin.

Eye drops of poorly soluble drugs are frequently formulated as suspensions. Bioavailability of suspended drug depends on the retention and dissolution of drug particles in the tear fluid, but these factors are still poorly understood. We investigated seven ocular indomethacin suspensions (experimental suspensions with two particle sizes and three viscosities, one commercial suspension) in physical and biological tests. The median particle size (d50) categories of the experimental suspensions were 0.37-1.33 and 3.12-3.50 µm and their viscosity levels were 1.3, 7.0, and 15 mPa·s. Smaller particle size facilitated ocular absorption of indomethacin to the aqueous humor of albino rabbits. In aqueous humor the AUC values of indomethacin suspensions with different particle sizes, but equal viscosity, differed over a 1.5 to 2.3-fold range. Higher viscosity increased ocular absorption 3.4-4.3-fold for the suspensions with similar particle sizes. Overall, the bioavailability range for the suspensions was about 8-fold. Instillation of larger particles resulted in higher tear fluid AUC values of total indomethacin (suspended and dissolved) as compared to application of smaller particles. Despite these tear fluid AUC values of total indomethacin, instillation of the larger particles resulted in smaller AUC levels of indomethacin in the aqueous humor. This suggests that the small particles yielded higher concentrations of dissolved indomethacin in the tear fluid, thereby leading to improved ocular bioavailability. This new conclusion was supported by ocular pharmacokinetic modeling. Both particle size and viscosity have a significant impact on drug concentrations in the tear fluid and ocular drug bioavailability from topical suspensions. Viscosity and particle size are the key players in the complex interplay of drug retention and dissolution in the tear fluid, thereby defining ocular drug absorption and bioequivalence of ocular suspensions.

Molecular Insights on Successful Reconstitution of Freeze-Dried Nanofibrillated Cellulose Hydrogel.

The diversity and safety of nanofibrillated cellulose (NFC) hydrogels have gained a vast amount of interest at the pharmaceutical site in recent years. Moreover, this biomaterial has a high potential to be utilized as a protective matrix during the freeze-drying of heat-sensitive pharmaceuticals and biologics to increase their properties for long-term storing at room temperature and transportation. Since freeze-drying and subsequent reconstitution have not been optimized for this biomaterial, we must find a wider understanding of the process itself as well as the molecular level interactions between the NFC hydrogel and the most suitable lyoprotectants. Herein we optimized the reconstitution of the freeze-dried NFC hydrogel by considering critical quality attributes required to ensure the success of the process and gained insights of the obtained experimental data by simulating the effects of the used lyoprotectants on water and NFC. We discovered the correlation between the measured characteristics and molecular dynamics simulations and obtained successful freeze-drying and subsequent reconstitution of NFC hydrogel with the presence of 300 mM of sucrose. These findings demonstrated the possibility of using the simulations together with the experimental measurements to obtain a more comprehensive way to design a successful freeze-drying process, which could be utilized in future pharmaceutical applications.

Determination of Residence Time Distribution in a Continuous Powder Mixing Process With Supervised and Unsupervised Modeling of In-line Near Infrared (NIR) Spectroscopic Data.

Successful implementation of continuous manufacturing processes requires robust methods to assess and control product quality in a real-time mode. In this study, the residence time distribution of a continuous powder mixing process was investigated via pulse tracer experiments using near infrared spectroscopy for tracer detection in an in-line mode. The residence time distribution was modeled by applying the continuous stirred tank reactor in series model for achieving the tracer (paracetamol) concentration profiles. Partial least squares discriminant analysis and principal component analysis of the near infrared spectroscopy data were applied to investigate both supervised and unsupervised chemometric modeling approaches. Additionally, the mean residence time for three powder systems was measured with different process settings. It was found that a significant change in the mean residence time occurred when comparing powder systems with different flowability and mixing process settings. This study also confirmed that the partial least squares discriminant analysis applied as a supervised chemometric model enabled an efficient and fast estimate of the mean residence time based on pulse tracer experiments.

Preparation and characterization of hot-melt extruded polycaprolactone-based filaments intended for 3D-printing of tablets.

Hot-melt extruded (HME) filaments are an essential intermediate product for the three- dimensional (3D) printing of drug delivery systems (DDSs) by the fused deposition modelling (FDM) process. The aim of this study was to design novel polymeric 3D-printable HME filaments loaded with active pharmaceutical ingredients (APIs). The physical solid-state properties, mechanical properties, drug release and short-term storage stability of the filaments and 3D-printed DDSs were studied. Physical powder mixtures of polycaprolactone (PCL), plasticizer and API were manually blended, extruded by a single-screw extruder, and printed by a table-top FDM 3D-printing system. The composition of PCL and arabic gum (ARA) enabled the incorporation of 20%, 30% and 40% (w/w) of indomethacin (IND) and theophylline (THEO) into the HME filaments. The uneven distribution of API throughout the filaments impaired 3D printing. The HME filaments loaded with 20% IND or THEO were selected for the further analysis and printing tests (the ratio of PCL, ARA and IND or THEO was 7:1:2, respectively). The IND filaments were yellowish, mechanically strong and flexible, and they had a uniform filament diameter and smooth outer surface. The filaments containing THEO were smooth and off-white. The 3D-printed tablets fabricated from IND or THEO-loaded filaments showed sustained drug release in vitro. The drug release rate, however, significantly increased by changing the geometry of 3D-printed tablets from a conventional tablet structure to an unorthodox lattice ("honeycomb") structure. Overall, the combination of PCL and ARA provides an interesting novel polymeric carrier system for 3D-printable HME filaments and tablets.

Continuous Pharmaceutical Manufacturing.

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Raman imaging of amorphous-amorphous phase separation in small molecule co-amorphous systems.

Many new active pharmaceutical ingredients (API) undergoing development have low permeabilities or low aqueous solubilities. However, the amorphous state is usually more soluble than its crystalline counterpart. The amorphous state has a higher Gibb's free energy, which can improve the apparent solubility but decrease the stability since the amorphous state tends to transform to the more stable crystalline form. Before recrystallization, a co-amorphous binary mixture's ingredients have to undergo a phase separation. The aim of this study was to obtain a better understanding of the amorphous-amorphous phase separation in co-amorphous binary mixtures and test the suitability of imaging Raman spectroscopy for detecting this phenomenon. To study the phase separation, we prepared three different 50:50 mass ratio binary mixtures of APIs: paracetamol-terfenadine, (PAR-TRF), paracetamol-indomethacin (PAR-IMC) and terfenadine-indomethacin (TRF-IMC). The binary mixtures were amorphized with melt-quenching and stored above their glass transition temperature (Tg) to monitor their phase separation. Thermal degradation was determined with a high performance liquid chromatography (HPLC) method to ensure that melt-quenching did not cause any thermal degradation of the molecules. Thermodynamic attributes (crystallization tendency, melting point (Tm) and Tg) were measured with differential scanning calorimetry (DSC) to ensure that the co-amorphous systems transformed to the amorphous state and remained amorphous after cooling and reheating. Phase separation was studied from the surface and cross-section (CS) with Raman imaging to examine if it occurred more on the surface than in the bulk. The Raman spectra were analyzed with principal component analysis (PCA) and Contour plots were produced from the PCA-score values to visualize concentration differences in the mixtures. The results showed that API vs API concentrations increased as a function of time in both surface and CS images before crystallization. This suggests that Raman imaging is a suitable technique to detect the phase separation phenomena in small molecule co-amorphous binary mixtures.

Converting a batch based high-shear granulation process to a continuous dry granulation process; a demonstration with ketoprofen tablets.

When one wishes to convert a batch based manufacturing process of an existing tablet product to a continuous process, there are several available strategies which can be adopted. Theoretically, the most straightforward way would be to proceed with the corresponding processing principles, for example to change a wet granulation (WG) batch process into its continuous WG counterpart. However, in some cases, the choice of roller compaction (RC) could be very attractive due to the notably simpler and inherently continuous nature of the RC manufacturing principle. The aim of this study was to examine a process conversion from batch based high-shear wet granulation (HSWG) to continuous RC manufacturing, without any significant formulation changes. An optimization of the formulation is often needed during the process conversion. However, our primary goal was to demonstrate the possibilities to perform this kind of process adaptation with minimal formulation changes. Furthermore, the effect of three different locations of lubrication feeding with two production rate levels was studied. An additional target was to identify possible over-lubrication with these manufacturing configurations, and to clarify which of these three possibilities steps produced a final product that conformed to the same quality requirements as HSWG tablets. Previously, the effects of lubrication only on compacted ribbons (Miguelez-Moran A.M, 2008) and final product with CDC (continuous direct compression) (Taipale-Kovalainen, et al., 2017; 2019) have been investigated. Here, the effect of lubrication on both ribbon and on final product was examined. No signs of over-lubrication were observed, but there was a clear effect of the feeding location of lubricant on the final product. On the basis of these results, it is concluded that in the future, if a good product/process understanding of the alternative manufacturing process with different techniques can be obtained, it will be possible to devise more flexible and effective ways to allow the pharmaceutical industry to switch from batch manufacturing towards CM.

The Comparison of Two Challenging Low Dose APIs in a Continuous Direct Compression Process.

Segregation is a common problem in batch-based direct compression (BDC) processes, especially with low-dose tablet products, as is the preparation of a homogenous mixture. The scope of the current work was to explore if a continuous direct compression (CDC) process could serve as a solution for these challenges. Furthermore, the principle of a platform formulation was demonstrated for low dose tablets. The combination of filler excipients and the API in the formulation used was suitable for direct compression, but also prone to induce segregation in BDC process. The CDC process was found to be very promising; it was shown that tablets with the desired quality parameters could be manufactured successfully with both of the APIs studied. Powder analysis indicated that the APIs display some fundamental differences in their physical properties, which was also reflected in powder mixture properties and, hence, eventually in processing. However, process parameters, especially mixer impeller speed, were not found to have any significant influence on end product quality. The study suggests that a CDC process can be a viable solution to resolve the challenges described. Moreover, manufacturing by using a universal platform formulation seems to be a feasible way for producing low-dose tablets.

Detection of amorphous-amorphous phase separation in small molecular co-amorphous mixtures with SEM-EDS.

Amorphicity is one possible way to increase the solubility of poorly water soluble drugs. However, amorphous solids are thermodynamically unstable and tend to recrystallize with material-specific kinetics. Crystallization is not the prime phenomenon in the whole process, although it is the easiest to measure. The primary phenomenon prior to the crystallization of glass is phase separation, the detection of which is very rarely reported among small molecular compounds. In the present study, a scanning electron microscope with energy dispersive X-ray spectrometer (SEM-EDS) was used to detect very early stage amorphous-amorphous phase separation in co-amorphous drug mixtures. Miscibility was calculated for five studied mixtures based on the Flory-Huggins method and four immiscible pairs and one partial miscible pair were selected for the laboratory experiments. Co-amorphous samples (n = 3) were prepared by melt-quench method and stored at the elevated temperature to induce the separation of amorphous phases. Each sample was stored at the same relative percentage temperature between glass transition temperature Tg and melting temperature Tm. Immediately after the sample preparation, the full amorphousness was verified with polarizing light microscopy. Before SEM-EDS analysis, the samples were fractured into two pieces and measurements were done from cross-section (from the bulk sample). All five pairs phase separated during two days of storage at the elevated temperature. The study proved that SEM-EDS was able to detect a very small phase separated regions in the amorphous sample, as amorphous-amorphous phase separation was detected in four out of five pairs. However, the surface roughness could affect the analysis and give a false indication of phase separation. SEM-EDS also supported calculation results, since every studied pair showed phase separation during study, as was predicted on the grounds of Flory-Huggins miscibility calculation.

Comparison of Traditional and Ultrasound-Enhanced Electrospinning in Fabricating Nanofibrous Drug Delivery Systems.

We investigated nozzleless ultrasound-enhanced electrospinning (USES) as means to generate nanofibrous drug delivery systems (DDSs) for pharmaceutical and biomedical applications. Traditional electrospinning (TES) equipped with a conventional spinneret was used as a reference method. High-molecular polyethylene oxide (PEO) and chitosan were used as carrier polymers and theophylline anhydrate as a water-soluble model drug. The nanofibers were electrospun with the diluted mixture (7:3) of aqueous acetic acid (90% v/v) and formic acid solution (90% v/v) (with a total solid content of 3% w/v). The fiber diameter and morphology of the nanofibrous DDSs were modulated by varying ultrasonic parameters in the USES process (i.e., frequency, pulse repetition frequency and cycles per pulse). We found that the USES technology produced nanofibers with higher fiber diameter (402 ± 127 nm) than TES (77 ± 21 nm). An increase of a burst count in USES increased the fiber diameter (555 ± 265 nm) and the variation in fiber size. The slight-to-moderate changes in a solid state (crystallinity) were detected when compared the nanofibers generated by TES and USES. In conclusion, USES provides a promising alternative for aqueous-based fabrication of nanofibrous DDSs for pharmaceutical and biomedical applications.

Measurement of residence time distributions and material tracking on three continuous manufacturing lines.

Over the recent decade, benefits of continuous manufacturing (CM) of pharmaceutical products have been acknowledged widely. In contrast to batch processes, the product is not physically separated into batches in CM, which creates a few challenges. Product release is done for batches that should have a uniform quality over time, materials need to be tracked along the line, and locations to reject product must be established. To enable these, the residence time distributions (RTDs) of all unit operations must be known. In this paper, three CM tableting lines, each employing a different granulation technique, were investigated. The RTDs of their main unit operations were characterized, utilizing different measurement techniques successfully. All of these RTD measurement techniques could have been performed in any of the lines. The differences were related to the techniques themselves. Overall, external tracer with in-line Near-Infrared detection or color tracer with video recording proved most usable techniques, with few limitations. The RTDs for full lines were calculated by convoluting the unit operation RTDs, which enables material tracking through entire lines. The lines exhibited both truly continuous and quasi-continuous unit operations. Quasi-continuous unit operations divide the material stream into lots that can be utilized for tracking and rejection.