Jennifer Markarian is manufacturing reporter for Pharmaceutical Technology.

Jennifer Markarian

	Twin-screw extruders, which have found commercial use in making amorphous solid-dispersions for improving drug solubility and in loading drugs into polymers that can be shaped into drug-delivery devices (i.e., via hot-melt extrusion), are also emerging as equipment for continuous wet granulation, as well as for continuous mixing of wet gelatin mass for soft-gel capsules. 

	The ongoing development and commercialization of continuous manufacturing for oral solid-dosage (OSD) pharmaceuticals is creating a growing opportunity for continuous granulation processes, and twin-screw granulation (TSG) is already being used in commercial continuous manufacturing. Some continuous systems, such as GEA’s ConsiGma and Glatt’s MODCON, for example, use TSG. Twin-screw extruders are available in models designed for pharma processing from manufacturers including Coperion, CW Brabender, Leistritz, Steer, Thermo Fisher Scientific, and others.

	A twin-screw extruder has a modular design, and different types of screw elements can be arranged on the screw shaft to optimize mixing and to allow ingredients to be added to the machine at multiple locations (i.e., part-way through mixing). The same machine can be used for wet granulation as for hot-melt extrusion, but the hot-melt extrusion process requires external barrel heating to raise the temperature above the melting point (or glass transition temperature) of the polymeric excipient, while granulation takes place at lower temperatures, below the excipients’ melting point or glass transition temperature. 

	The primary benefit of TSG is that the extruders are intended for continuous manufacturing, notes Dirk Leister, technical marketing manager, process and pharma extruders for Thermo Fisher Scientific. “Extruders are time-based production-the same equipment can be used for different amounts of material. One can potentially use the same extruder for R&D as for production, by simply running longer. Or, the process can be scaled up to a larger extruder for higher throughput.” Thermo Fisher’s Pharma 11 (11-mm diameter) and Pharma 16 (16-mm diameter) twin-screw extruders are designed for both R&D and production. These extruders can be set up for both granulation and hot-melt extrusion, using conversion kits for the necessary hardware modification. Thermo Fisher recently introduced the Thermo Scientific Pharma 24 TSG, a 24-mm diameter twin-screw extruder dedicated for twin-screw granulation with a throughput rate of up to 70 kg/h. The extruder can be either integrated into a continuous production line or used as a standalone instrument for project development or small-scale production using either wet or dry granulation (1). The extruder has a 40:1 ratio of length to diameter that offers flexible adaptation for processing length, screw setup, and introduction of ingredients into the process. Minimal downtime is needed for cleaning, and online monitoring facilitates detection and segregation of out-of-specification product.

	In addition to flexibility of scale, continuous processes offer the potential for improved process control compared to batch processes and for on-line, real-time monitoring. The twin-screw extruder is considered a small-volume continuous mixer, and it creates a more uniform mixing history for more homogeneous distribution of drug, excipient, and binder (2).

	“Consistency is a big advantage,” notes Michael Thompson, professor in the department of Chemical Engineering at McMaster University in Ontario, Canada. “TSG can reduce lot-to-lot variation of the granulated product and reduces reliance on a specific seasoned operator in order to get the correct product quality by knowing when to stop the batch mixer.” TSG may need less binder or less water to produce equivalent granules to batch systems, which could reduce production cost. 

	How the primary variables in the TSG process affect product properties is relatively well understood at this point, although each formulation would need to be optimized. “Just about any formulation based on known pharmaceutical ingredients can be made into granules so long as the formulator has a working knowledge of the different techniques to ‘wet out’ the powders,” says Thompson. “I would always recommend to a company to study the binder-to-powder needs in each new formulation as if starting from scratch, but the initial machine setup and operating conditions can be treated as relatively generic.” Scaling up from a small laboratory machine to larger production equipment poses more challenges, however. “One can’t truly know a process until it has run for several hours, with the machine heated up by mechanical rotation and the feeders having gone through several fill cycles to fully learn the bulk density of the process’s ingredients,” cautions Thompson. “A product made from a 20-minute lab trial doesn’t necessarily translate to a continuously operated production process.”  

	Process analytical technology (PAT) is a crucial part of understanding and controlling a continuous process, and in-line measurements are used in advanced process control strategies and in quality-by-design studies. Innopharma Technology’s Eyecon

, for example, is a direct optical imaging system that captures images of the granule particles and analyzes them in real-time. “In continuous processing, the particle analyzer can be used to monitor particle size after a twin-screw, a continuous drier, or a mill, says Chris O’Callaghan, senior product manager at Innopharma.”

	In a continuous system, data are needed for both feed-forward and feed-back control to maintain key parameters and attributes as close to an ideal setpoint as possible, despite some variation in the inputs and outputs of each process stage. “While feed-back control is critical in ensuring that deviations do not continue to grow unchecked, feed-forward control can enable compensation of upstream variations in subsequent processing steps to ensure that variations introduced in early stages can be partially or fully corrected in the final output material,” explains O’Callaghan.

	“By measuring particles after granulation, deviations in the measured size can be used to alter parameters of the twin-screw (e.g., liquid addition and screw speed) to correct for the variation in particle size, while the speed of a downstream milling step may be temporarily increased or decreased to more finely or coarsely mill the granulate currently entering, thereby maintaining a more consistent output particle size.”

	More work is needed to further develop different types of PAT for TSG, says Thompson. In the McMaster University laboratory, researchers are looking at ultrasonic acoustic sensors to see what information about properties exiting the twin-screw granulator, such as granule size and moisture content, can be measured. Eventually, the researchers hope to also identify technology that can monitor incoming powders, which can also have a significant effect on end properties. For example, the size of a binder particle is known to affect granule size in hot-melt granulation, but a supplier of binder material might not measure or report binder particle size or changes in grinding because they wouldn’t affect chemical properties, notes Thompson. It would be difficult to track a problem with off-spec granules back to the feed materials, and a formulator might mistakenly assume something inside the extruder had gone wrong. 

	Sensors that are already built into extruders can also be used as PAT signals. The torque of the extruder drive motor, for example, should stay constant duringsteady-state processing, and can be used in process control, notes Leister. 

	Twin-screw extruders can also be used for continuous processing of gel mass used in casings for soft-gel capsules. In April 2018, Leistritz introduced a twin-screw extrusion (TSE) system to continuously mix and condition gel-mass formulations. The system, which uses a ZSE-LS model twin screw extruder, replaces batch mixing for gel-mass R&D and production processes, and the continuous process is inherently more consistent and repeatable, says the company. Continuous, real-time monitoring of the TSE process parameters ensures product quality.

	“The short residence time inherent with the TSE process section helps avoid degradation of the gel-mass,” explained Leistritz (3). “It is estimated that the residence time in the TSE process section is less than two to three minutes. … Raw ingredients (gelatin, starches, water, plasticizer, and other additives, such as colorants and flavors) are sequentially metered into the extruder process section where they are heated, hydrated, mixed, and pumped to an intermediate filling station.  The intermediate filling station refills two spreader boxes to form thin webs on chilled rolls, which are integrated with die rollers and liquid injection apparatus to produce soft-gel capsules.”  

	The food and polymer industries successfully transitioned from batch to continuous processing using twin-screw extruders fifty years ago, and use of TSG is expected to continue to evolve in the pharma industry, say experts (2). 

	1.    Thermo Scientific, “New Twin-screw Granulator Helps Maximize Throughput in Continuous Drug Manufacturing,” Press Release, June 25, 2018.

	2.    N. Kittikunakorn, et al., “Processes, Chllenges, and the Future of Twin-Screw Granulation for Manufacturing Oral Tablets and Capsules,” AAPS News Magazine, March 2018.

	3.    Leistritz, “Leistritz Introduces a Continuous Twin Screw Extrusion System That Replaces Batch Mixing for Gel Mass R&D and Production,” Press Release, April 16, 2018.

Articles

A twin-screw extruder can be used as a continuous wet granulator or as a continuous mixer for a wet gelatin mass. 

Considering Twin-Screw Extruders for Continuous Mixing and Granulation

	Orally inhaled and nasal drug products (OINDPs) are formulated as either solutions, suspensions, or dry powders that are delivered using devices such as nebulizers, metered dose inhalers (MDIs), or dry powder inhalers (DPIs). One of the critical quality attributes of an inhaled drug product is the amount and size of foreign particles in the product. “Foreign particles-including glass, stainless steel, or different types of polymers-can come from any step in the manufacturing process or from the source materials,” notes John Bak, PhD, principal scientist at PPD Laboratories’ GMP Lab. Manufacturers must develop appropriate tests to identify and measure these particles so that they can be eliminated or maintained below a certain level. 

 spoke with Bak to learn best practices for developing these tests.

 guidance documents are used to develop appropriate tests for foreign particulate matter in inhaled products?

 Current guidance documents that are available include:

 <5> Inhalation and Nasal Drug Products–General Information and Product Quality Tests (1)

Nasal Spray and Inhalation Solution, Suspension and Spray Drug Products-Chemistry, Manufacturing and Controls Documentation

Metered Dose Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug Products

	Although the requirement to do particulate matter testing is included in these documents, no techniques or guidelines for specifications are given. It is up to the applicant to develop and justify the choices made.

 <788> Particulate Matter in Injections (4) and its specifications are specifically for parenteral products and are not appropriate for OINDPs. The testing procedures can be used to help develop an appropriate test, but not the specifications.

 provide little guidance, there have been some position papers published to fill the gap. Two papers by Blanchard et al., ‘Foreign Particles Testing in Orally Inhaled and Nasal Drug Products’ (5) and ‘Best Practices for Managing Quality and Safety of Foreign Particles in Orally Inhaled and Nasal Drug Products, and an Evaluation of Clinical Relevance’ (6), describe the types of tests that can be done and a rational means of setting product specifications specifically for OINDPs.

	The means of setting specifications proposed in the paper (5) is to use the US Environmental Protection Agency National Ambient Air Quality Standards (NAAQS) for particles with < 10 µm aerodynamic diameters. A small percentage (1–5%) of the allowable exposure is suggested for the maximum daily exposure due to the use of the product.

	The first article (5) also has a survey of the commonly used techniques for testing for particulate contamination along with each technique’s strengths and weaknesses.

What factors should be considered when deciding upon a testing strategy?

 Based upon the recommendations of these two articles (5,6) and our experience, when looking at a strategy to test a product under development, the device used for delivery (e.g., nebulizer, MDI, or DPI), the location of action (e.g., nasal or pulmonary), the state of the formulated drug (e.g., solution, suspension, or dry powder), and the testing strategy (e.g., delivered dose or extracted formulated drug product procedures), all impact choices that are made as far as the technologies used, the test method validation strategies applied, the robustness of the resulting procedure, and the cost of the ongoing testing for stability and release.

	The first step in putting together the strategy is to harvest the particles from the drug product, drug substance, excipients, and delivery devices and analyze them using microscopic techniques that allow chemical identification, such as Fourier Transform infrared (FTIR) microscopy, Raman microscopy, and scanning electron microscopy with energy dispersive X-ray probe (SEM/EDX), to elucidate what the common contaminants are and identify their sources so they can be eliminated or reduced in the manufacturing process. In addition, a toxicological assessment can be performed to ensure that no particularly harmful materials are present. Special consideration is given to the < 10 µm particles because they are respirable and therefore present the highest risk from exposure.

	Understanding the particulate profile and having it be reasonably stable from lot-to-lot demonstrates control of the manufacturing process with respect to particulate contamination in the drug product. When this level of understanding is established, the use of faster and less-expensive techniques to monitor the level of particulate contamination is warranted. Light obscuration is the most common technique used for monitoring.

What are the considerations for choosing to collect and analyze particles using the delivered-dose technique or analyzing the drug product extracted from the device or container?

 The delivered-dose technique sounds better because it simulates the actual exposure the patient receives. These techniques, however, tend to be difficult to implement because of their poor precision, which requires higher-skilled analysts and thereby increases the cost of testing. In addition, the incidence of atypical and out-of-specification result investigations is increased with the associated delays. If a justification can be made to use material extracted from the device or container, the routine testing for stability and release is typically more robust and trouble-free.

	Delivered-dose techniques for particulates often can be created by modifying methodology used in delivered-dose uniformity testing. A sufficient amount of material is collected, and then any solid drug substance or solid excipients are dissolved using an appropriate particle-free solvent. If no other solids are present, a compatible suspending solvent is added instead. The solvent is then well mixed to suspend the captured particulate contamination and tested by light obscuration or other techniques. This approach allows the use of the same or similar devices that analytical teams are used to working with, for instance, those described in 

 <601> Inhalation and Nasal Drug Products: Aerosols, Sprays, and Powders

 <1601> Products for Nebulization-Characterization Tests (8).

	When extraction techniques are used, the testing is usually simplified. For nebulizers, the blow-filled vials are opened and the liquid pooled. The pool is then tested by light obscuration or another technique. For DPIs, the powder is extracted as appropriate. For instance, blister strips are cut open and the powder pooled. The powder is then dissolved in an appropriate solvent. The remaining particulate matter is then suspended in the solution, and it is tested. For MDIs, the material is extracted by cutting open the frozen can or by dispensing into a container that catches the drug product. If needed, any solid excipients or drug substance are then dissolved in a particle-free solvent. The resulting solution has the particulates suspended and is then tested.

	Light obscuration typically is preferred for analyzing the resulting test solutions. If visible light microscopy is used, the solutions are filtered, and the remaining particulate contamination is counted. The magnification should be set high enough that the < 10 µm particles are clearly visible. Techniques based on 

 <788> often undercount particles in the 2–5 µm range because at 100x magnification specified in 

 <788> (4), these particles are difficult to see against the background of a filter membrane. Light obscuration testing, on the other hand, has good sensitivity to particles in the 2–5 µm range.

What are the most significant challenges in developing appropriate tests for foreign particulate matter?

 Typically, the most challenging part of developing the testing is to find a solvent that dissolves the drug substance and the excipients at the same time, while minimizing interfering artifacts like microbubbles. The second most significant challenge is cleaning the equipment in a particle-free environment to control laboratory contamination of the samples.

What is typically required for validating these analytical methods?

 We typically recommend that the < 10 µm particles be validated as a quantitative impurity test, since they present the highest risk because they are respirable. The range of analysis is demonstrated from the limit of quantitation to 120% of the product specification along with any demonstrations of method robustness indicated from the method development. The ≥ 10 µm and ≥ 25 µm particles also can be validated in the same manner, or be shown to have impurity-limits compliance, because they present less risk and are monitored more from a product quality standpoint than a safety risk.

 40 Supplement 2 <5> Inhalation and Nasal Drug Products

General Information and Product Quality Tests (2017).
	2.     FDA, 

Nasal Spray and Inhalation Solution, Suspension and Spray Drug Product-Chemistry, Manufacturing and Controls Documentation

 40 Supplement 2 <788> Particulate Matter in Injections (2017)

 21 (12) 2137-2147 (2004).
	6.     J. Blanchard, et al. 

Performance Quality Tests (2014).
	8.     USP, 

 40 Supplement 2 <1601> Products for Nebulization-Characterization Tests (2017).

When choosing a test method to identify and measure particulate matter in inhaled drug products, the delivery device and drug form should be considered.

Developing Test Methods for Foreign Particulate Matter in Inhaled Drugs

	Biopharmaceutical manufacturing companies have an opportunity to participate in the latest industrial revolution, which is being called Industry 4.0. In this digitalized paradigm, machines collect process and product data and communicate with other machines via the Industrial Internet of Things (IIoT).  Using artificial intelligence (AI) or machine learning, machines can even use these data to improve processes without human intervention, although how to use AI in a regulated, GMP-compliant space is yet to be resolved. In fact, in many cases, the bio/pharma industry is still transitioning from manual systems to automation (i.e., “Industry 3.0”) (1).  Next-generation technologies are already providing more data that, along with advanced analytics and modeling technologies, can help increase process understanding and be used for advanced process control and efficiency improvements. 

	One of the hallmarks of Industry 4.0 is “big data” collected by equipment sensors and process analytical technologies (PAT). These manufacturing data can be used to analyze what is happening in a process and why, to predict what will happen in response to given changes, and-using simulation algorithms-to optimize processes, explained Lennart Eriksson and Chris McCready of Sartorius Stedim Data Analytics (2). Eriksson calls this higher level of simulation and optimization “prescriptive data analytics.” Others call it process modeling, and some give the name “digital twin” to a process model that is a virtual copy of the actual process. Modeling has become more sophisticated, enabling process designers to run virtual experiments and gain a greater understanding of their process before actually running it. 

	Digital twins are also being used to predict maintenance and downtime more accurately; plan for process improvements, such as the replacement of components; and prepare alternative plans in case of malfunctions or disturbances, all without interrupting manufacturing, explains Billy Sisk, Life Sciences industry leader at Rockwell Automation. 

	Sanofi, for example, is bringing digital transformation to its biopharmaceutical manufacturing facilities. Each of the company’s digital plants will have a digital twin connected directly to the sensors and data in the physical plant. “The data flows to these digital twins, giving managers a real-time view into the plant’s operation. Simulation on the model provides the level of manufacturing modularity and future flexibility required to support personalized medicine,” Sanofi said in an October 2017 press release (3). At its Geel, Belgium biologics manufacturing facility, sensors generate more than one billion data points in every manufacturing cycle. “By analyzing this data, potential deviations can be spotted and swiftly corrected. Yield is improved with adaptive process control strategies, and downtime is reduced by optimizing maintenance activities and shifting to predictive maintenance, based on equipment monitoring, which will increase overall output,” reported the company.

New technologies that allow better process control are improving efficiency and reducing waste in biopharma manufacturing by automating tasks or assisting operators to improve manual task handling. GE Healthcare’s XDUO, for example, is a new “smart” mixer for buffer solutions that continuously measures pH and performs automatic titration using acid and base pumps. This solution creates tighter control and saves a significant amount of time compared to manual titration, notes the company. 

The use of PAT to measure critical quality attributes in real-time is well-established in upstream bioprocessing, but more research is needed to apply it to downstream development and manufacturing (4). Work is underway: multi-attribute methods (MAM) that use mass spectrometry as PAT, for example, are being developed in cooperation with FDA’s Emerging Technology Team (4,5).

	As biopharmaceutical manufacturers transition to Industry 4.0, they should consider the requirements for changes to processing equipment, such as the automation of bioprocessing skids, and increased data security. 

The moveable, skid-mounted biopharmaceutical manufacturing equipment in use today has different requirements for automation than fixed equipment. Fixed equipment typically uses a distributed control system (DCS). Stand-alone skids, however, typically use programmable logic controllers (PLCs), which work well for isolated equipment but are more complex to integrate into supervisory systems, such as a DCS or a manufacturing execution system (MES), explains Bob Lenich, Global Life Sciences director at Emerson. To solve this problem, Emerson’s new DeltaV PK Controller is a DCS controller for skids that are self-contained, like a PLC, but can be more easily plugged into a plant-floor automation system, such as Emerson’s Delta V DCS (for coordinated operations in real-time with alarming, automated closed loop control, an integrated user interface, and historical data collectionthat a DCS provides) or into a MES (for stand-alone pieces of equipment that only need limited direction on what to produce). 

	“In the near future, we anticipate that there will be more of two types of skids: mobile and movable skids, with the difference being that mobile skids are moved much more frequently,” says Lenich. “What’s missing today is a tracking mechanism to identify the physical locations of mobile and movable skids. Many of our customers are interested in using [radio-frequency identification] RFID tags as a potential solution for mobile skids because they need constant tracking. In contrast, moveable skids are repositioned less frequently, and most end users would be fine with something simpler like a barcode or QR code that doesn’t require constant tracking.”

	The use of skids and super-skids with single-use (i.e., disposable) components presents additional automation challenges, notes Torsten Winkler, lead of the Life Sciences Center of Excellence in EMEA for Honeywell Process Solutions. “It is important to identify single-use equipment and, more importantly, that all are connected properly. Automation systems can continuously monitor when skids are connected, but there are additional challenges with identifying the disposable parts,” says Winkler. 

	Single-use facilities and skid-based equipment require a significant amount of manual set-up to connect all the equipment pieces. Producing a batch in a single-use bioreactor, for example, might require up to 900 individual connections, and any mistakes can cause the batch to fail, notes Sisk. Graphical aids can help operators visualize the instructions, and equipment recognition and verification systems can be integrated into the control platform to track and confirm equipment placement. For example, the control system could be set up to require the operator to scan a barcode on a disposable part, which would become part of the batch record.

With the increasing connectivity of systems communicating over the IIoT, data security has become even more important. Systems must use modern protocols that have adequate security. Another best practice is to segregate non-GMP business data from GMP process data using an “industrial demilitarized zone” in information technology systems, note experts. Mobile connection, such as having access to process data on mobile phones or tablets, is an issue manufacturers must consider. The technology is available, but it must be managed to maintain data security. 

	“Organizations have adopted ‘safety cultures’ for years, but now they need security cultures,” says Lenich. Just as a safety culture uses various programs to make the importance of safety visible to everyone in the organization, a security culture would similarly highlight that security is everyone’s responsibility and make security best practices a regular part of day-to-day priorities, he explains.

	2.    L. Eriksson and C. McCready, 

	3.    Sanofi, “The Digital Plant: From Collaborative Robots to Virtual Reality,” Press Release, Oct. 24, 2017.

	5.    R. Deshpande, “Progressive Technologies to Realize the Vision of Advanced Biomanufacturing,” presentation at IFPAC 2018 (North Bethesda, MD, 2018).

Modern technologies, including Industry 4.0 and the Industrial Internet of Things, offer opportunities to increase biopharmaceutical manufacturing efficiency.

Industry 4.0 in Biopharmaceutical Manufacturing


	Vol. 30, No. 7
	July 2018
	Pages: 33–34

	When referring to this article, please cite it as J. Markarian, "Testing Foreign Particulate Matter in Inhaled Products," 

The delivery device and drug form should be considered when choosing a test method for identifying and measuring particulates in inhaled drug products.

Testing Foreign Particulate Matter in Inhaled Products

	In aseptic and sterile drug fill/finish and inspection, automation can reduce or eliminate operator intervention, which reduces contamination potential and improves efficiency. Traditional, one-axis automation works well for high-speed processes, but multi-axis robotic arms offer repeatability with the flexibility to easily change the process or the type of container being filled. Technology advances have made robotic systems suited for aseptic processing and easy to operate. 

	While the pharma industry may be more familiar with robotics used in packaging operations, advances in technology, such as “near zero particle generating designs and complete tolerance to 

biodecontamination cycles with gaseous hydrogen peroxide” have made robotics compatible with aseptic manufacturing, explains Josh Russell, director of business development and technical sales at AST. 

	Today’s robotic systems are also easily reprogrammed for changes after the system is set up, without requiring a programming specialist on staff, say suppliers. Some platforms, such as those from AST and Vanrx Pharmasystems, are recipe-driven, and an operator inputs the recipe information-such as the type of container and volume to be dispensed-through a touchscreen interface. 

	Flexible and repeatable filling are becoming more crucial due to changes in the biopharma industry, notes Chris Procyshyn, CEO at Vanrx Pharmasystems. “The focus on targeted therapies for smaller patient populations creates a challenge for companies to produce a higher number of smaller batches into different container formats. Biologics, plus new modalities such as cell and gene therapies, are more complex and process intensive. The use of robotics is growing in response to these changes,” explains Procyshyn. 

	A potential drawback to robotic systems is running speed. “These systems aren’t yet designed to run 500,000 unit batches or produce 100 million units a year. But that’s not where the demand is anyway, and conventional systems weren’t meeting the need for small batch flexibility,” says Procyshyn. “If a company is making high-value drugs into multiple formats or sizes, they want a robust, repeatable process that reduces risk. That happens best with a closed robotic filling system.”

	“Traditional aseptic systems are proving to not be well suited to address these personalized therapeutic products because they lack the ability to efficiently produce low-volume, high-mix batches,” agrees Russell. “However, robotic systems that have recipe-driven operation and the ability to process syringes, vials, and cartridges on a single platform are an ideal solution.” In addition, more companies are finding that robotic systems give them the flexibility, ease of use, and sterility assurance they need to replace manual filling of clinical batches. 

	“From a staffing standpoint, automating these tedious and repetitive processes reduces the risk associated with precise dosing and employee fatigue, and automation can reduce scrap and re-work [compared to manual methods],” adds Walter Langosch, Global Business Development director at ESS Technologies. For example, the company’s TaskMate robotic syringe filler/capper, which was introduced in December 2016, replaces manual methods and can fill and cap up to 15 syringes per minute. 

	“We clearly see a high demand for robotics in aseptic filling,” says Sebastien Schmitt, Robotics division manager, North America, at Stäubli. One growing use is in automated compounding pharmacies in hospitals, in which robots can be programmed flexibly to produce drugs on demand, adds Schmitt. Robotics provide consistent performance, with less equipment inside an isolator, hence, reducing risk and facilitating maintenance. 

	Steriline Robotics, a spin-off company created by Steriline with Milan Polytechnic, produces aseptic filling machines primarily used in hospitals and in pharmacy compounding. “The machine we developed uses two robots cooperating to help pharmacies in targeted drug reconstitution for each patient, according to their specific prescription,” notes Federico Fumagalli, chief commercial officer at Steriline. 

	Steriline is focused on “zero-loss” aseptic filling systems that aim to increase production efficiency and reduce the waste associated with rejects using traditional automation. Their robotic system can, for example, identify a filled vial that is missing a stopper and send it to be stoppered during a second cycle, preventing the loss of that vial, says Fumagalli. Steriline’s systems run faster than some other robotic systems due to their artificial-vision system, reaching a filling speed of almost 100 vials/min, he says. 

	Nested vials have been available for more than five years, and the technology to optimally handle vials in this format is now catching up, says Russell.  In AST’s ASEPTiCell and GENiSYS aseptic filling and closing machines, six-axis articulated robots perform all the critical aseptic operations, including removing the tub from its sterile overbag, opening the tub, and denesting the containers (i.e., vials, syringes, or cartridges). After filling, the robot closes the containers almost immediately, resulting in less time that the sterile drug product is exposed to the aseptic environment. “The system is completely closed. It eliminates operator intervention and allows a completely aseptic fill,” says Russell. Depending upon the application requirements, the system can be used inside a restricted access barrier system (RABS), isolator, or laminar airflow hood. 

	The robotic system allows a streamlined deck that is easier to clean than traditional automation, explains Russell. Another advantage over fixed automation is that the robot has various pre-programmed “maintenance positions” where it is ergonomically positioned for operators to access the disposable fluid path and to perform other necessary aseptic tasks without violating basic “first air” principles. In addition, the robotic system enables a real-time fill-weight check. AST’s machine uses an in-process feedback loop to the filling pump for closed-loop control. In addition to the commercial-scale ASEPTiCell i100 line, a laboratory-scale line GENiSYS was introduced in October 2017 for small-scale filling for R&D (e.g., stability studies), for clinical production, and also for small-scale commercial production, such as cell and gene therapy. 

	A common use of robots in aseptic processing is to use a robot to replace certain operations, such as removing the cover from a nested container tub. Entirely rethinking the aseptic filling process, however, allows manufacturers to take full advantage of robotics. Procyshyn explains, “First, robots provide a highly repeatable production process. Using recipe-driven processes, they are able to produce multiple drug products at equally high quality levels into different containers. Second, robotic systems are agile and change quickly between vial, syringe, and cartridge formats. Robots can handle every type of container the same way if the containers and closures are nested, so there are limited change parts. Without traditional electro-mechanical components complicating the machine’s interior, decontamination cycles are much shorter too. Third, robots allow the design of gloveless isolators. A gloveless isolator can run much cleaner than a conventional isolator and be completely closed. [There are] no mouse holes, conveyors, glass-to-glass contact, or sortation bowls, which all create risks to the batch.” 

	Procyshyn notes that Vanrx Pharmasystems SA25 Aseptic Filling Workcell gloveless isolator technology is based on a standard design, which allows filling operations to be built faster than before (with lead times of less than a year) and in less space. The workcell design provides agility, with the ability for one machine to fill multiple container formats with minimal changeover times. “Gloveless isolator technology provides better aseptic assurance than conventional isolators or RABs,” adds Procyshyn. The workcells are entirely closed to the outside environment, and they also use single-use product-contact materials. 

	Contract development and manufacturing organization (CDMO) FUJIFILM Diosynth Biotechnologies announced in a May 23, 2018 press release that it was investing in the SA25 Aseptic Filling Workcell gloveless isolator technology as it expanded its gene therapy and viral vaccine fill/finish capabilities to provide services in support of late-phase candidates and commercial supply (1). Fujifilm highlighted the advantages of automated environmental monitoring and no glass-to-glass contact.  

	Vanrx Pharmasystems recently introduced a smaller-scale gloveless isolator system, theMicrocell vial filler, designed for rapid scale-out of personalized medicines, such as cell and gene therapies and immuno-oncology products, from clinical through commercial manufacturing. (2). The system replaces manual filling and small-volume filling machines in biosafety cabinets, RABS, or isolators, says the company. The Microcell can fill 2R-50R nested vials with toolless changeover and decontamination in 15 minutes. 

	PTI–Packaging Technologies & Inspection’s new robotic inspection system uses the company’s MicroCurrent High Voltage Leak Detection (HVLD

) to test the container closure integrity (CCI) of prefilled syringes. The E-Scan 665 tester is a fully automated system in which a six-axis robot picks and places rows of syringes from the nested trays into the HVLD

and returns good product to the tray and faulty product to a reject tray. The inspection equipment company chose to use robotic actuations for the automated system because of the complex and delicate component handling required, says Oliver Stauffer, CEO of PTI. Robotic handling can have throughput limitations and may not work for higher speed processes, he notes, but it is suitable for this application, in which a tray of syringes is checked in less than three minutes. “Robotic systems provide flexibility in handling and adjusting for different format types and sizes. Technical requirements can often be achieved by simply adjusting positioning with the same end-of-arm tool,” says Stauffer.

	Stäubli’s TX Stericlean series was the first robot introduced in 2008 for aseptic production areas, says the company, which reports that its new TX2 line of collaborative robots (i.e., cobots) are also a first for aseptic processing. The use of cobots allows glove boxes to be located next to the robot, so that the operator can intervene in the isolator without stopping the production process. The TX2 robots are Industry 4.0-compatible, in that they can collect production data and are capable of communicating with each other, reports the company.

	1.    Fujifilm USA, “Fujifilm Announces the Expansion of Its Fill Finish Services to Include Late Phase and Commercial Production of Gene Therapies and Viral Vaccines to Become an End-to-End Solutions Provider,” Press Release, May 23, 2018.  

http://www.fujifilmusa.com/press/news/display_news?newsID=881420

	2.    Vanrx, “Vanrx Microcell Wins INTERPHEX ‘Best in Show’ Award,” Press Release, April 18, 2018. 

https://vanrx.com/vanrx-microcell-wins-interphex-best-show-award/

Automated robotic arms manipulate nested trays and containers inside closed aseptic filling systems.

Robotics Solve Aseptic Filling Challenges

Subvisible particles (SVP) are a concern for biologic-based drugs. Some technologies can detect but not identify particles. “Knowing that subvisible particles are present-without seeing what they are-can only take you so far. Without an image of the impurity, it may be difficult to figure out where the problem originates,” notes Kent Peterson, CEO of Fluid Imaging Technologies. 

spoke with Peterson about using imaging to identify subvisible particles in parenterals.  

In what situations in bio/pharma manufacturing would a manufacturer need to perform imaging for SVP?

SVP are either foreign contaminants or undesirable byproducts of the therapeutic molecules, and they may cause adverse effects and compromise patient safety. SVP can cause immunogenicity, and a SVP count that exceeds the parameters set by the 

	Biopharma companies analyze their product from formulation development all the way to patient administration. Formulation scientists can use the imaging capabilities of flow imaging microscopy to study the behavior, including stability and compatibility, of the therapeutic formulations. 

	SVP may form or be introduced into the formulation at any point during manufacturing or post-manufacturing operations. Research has shown that equipment used during manufacturing, including filters, filling pumps, and tubing, can shed foreign particles into the product. Additionally, particles have been found to form resulting from physical stresses during post-filling operations. Storage containers, such as vials and syringe components, have been found to shed during shipping and storage (1,2). Additionally, agitation during shipping, dropping the syringe or vial, or sending the intravenous (IV) bladder through a pneumatic tube (common in many hospitals) can result in fluid flows and cavitation, which can initiate particle formation/protein aggregation. Exposure to light can cause protein degradation. Extreme temperature changes and accidental freeze/thaw cycles can induce particle formation and/or protein aggregation, and improper IV dilution can cause particle formation. Research has shown that nanoparticles can serve as nucleation points for protein agglomeration when exposed to mechanical stresses, such as agitation during filling pump operation, and can result in a therapeutic unsafe for patient use (3,4). 

	Regular analysis throughout the manufacturing process can determine the point at which SVP form or are introduced, and analysis following post-fill operations can verify the quality of the product administered to the patient. Flow imaging microscopy enables the user to analyze the morphology of the particulate load, identify the SVP and their likely source, and remediate the issue.

	For example, a monoclonal antibody from a well-known drug manufacturer was forming visible particles during the final filling operation. Using flow imaging microscopy, the manufacturer identified that metal shards were being introduced to the product during filling operations. They linked the steel shard shedding to one pump in particular. The ability to see morphological characteristics enabled the manufacturer to identify the particle, target its source, and resolve the problem.

	In addition to analyzing SVP during manufacturing and post-manufacturing operations, the manufacturer would also need to perform particle analysis following a recall. 

requires that the product meet the particle size and count requirements using light obscuration (LO) or membrane microscopy as means of particle analysis. While both methods are acceptable, LO is the most commonly used method for its rapid results and ease of use, compared to standard membrane microscopy. However, LO does not capture, detect, or report on particle morphology other than particle size. It also often fails to detect small particles. Although particle size and count, as provided by LO, are important parameters to measure, subtle morphological cues can be used to identify the source of the SVP and determine whether it is intrinsic, extrinsic, or inherent based on its identity, and from there determine the point in the process it was introduced.  

What are the current industry regulations and how can this technique be used to comply with them?

Industry regulations for protein therapeutics continue to be modified and adapted as our understanding of proteins and our detection technology advances. When 

<788> was published, it required only the enumeration of particles greater than or equal to 10 µm and greater than or equal to 25 µm using LO or standard membrane microscopy (5). Since the publication of 

<788>, however, technology has advanced, and our understanding of proteinaceous particles has deepened. 

<787> was later published to serve as a complementary regulation to 

<788>, allowing smaller sample volumes to be used for analysis (6). 

<1787> considers our current understanding of particles in protein therapeutics, the dangers of foreign particles and poor characterization, and the inadequacies of using LO as the only characterization method (7). LO only provides particle count and size distribution. It is unable to identify the particle and determine whether it is extrinsic, intrinsic, or inherent. Therefore, 

<1787> recommends alternative methods, including flow imaging microscopy, for improved particle characterization and identification of foreign contaminants in effort to improve product safety.

	While LO or standard membrane microscopy analyses are required per 

regulations, flow imaging microscopy is recommended as an additional, orthogonal analysis for method verification and enhanced particle characterization. Particle morphology, captured by images, can be used to determine whether the SVP is inherent, intrinsic, or extrinsic; identify its source; and remediate and refine manufacturing and post-manufacturing operations. 

How does dynamic imaging particle analysis allow identification of subvisible particles?

Flow imaging microscopy images and analyzes particles ranging in size from 300 nm to 100+ µm suspended in a fluid in real-time. A liquid sample is introduced into the instrument and pulled through the flow cell using a micro-syringe pump. Objectives of varying magnifications can be installed to optimize imaging particles of various sizes. The flow cell is positioned between the camera and the illumination source. A strobe illumination is used to ‘freeze’ the moving particles in space and capture a clear image of each particle as the sample travels through the flow cell. As the images are captured, intelligent analysis software enumerates particles, analyzes the particulate populations, and measures over 40 different physical parameters from each particle image. Such parameters include length, width, area, diameter, circularity (Hu), elongation, convexity, and many more.

	Software filters based on morphological measurements can be built and used to distinguish between particle types (such as silicone oil droplets, bubbles, protein aggregates, glass shards) and enable analysis of each subpopulation. Size bins can be created to analyze particles in specified size ranges, such as the recommended 2–10 µm, and the required greater than or equal to 10 µm and greater than or equal to 25 µm. These images are then visually inspected by the user to identify the likely origin of the particle. Each particle image is saved to a user-built reference library and used to improve the software’s pattern recognition algorithms and improve particle identification.

	All particle types within the size range of 300 nm to 100+ µm can be analyzed using flow imaging microscopy. Unlike LO, flow imaging microscopy’s unique light and dark thresholding technology ensures that particles are detected, imaged, and analyzed, regardless of their opacity. Light obscuration instruments rely on the opacity of the particle to generate a shadow. The shadow produces a signal, correlated to the size of the shadow, from which particle size is inferred. Therefore, if the particle and its fluid medium have low contrast, the particles will not produce a strong enough shadow to be detected. This is particularly common with protein aggregates, which are transparent to semi-transparent in nature. Therefore, protein aggregates, due to their transparent nature, often fail to be accurately analyzed and even go undetected when analyzed using LO instruments. 

	Until recently, flow imaging microscopy could only analyze particles as small as 2 µm. The combination of a blue LED light and patented oil immersion technology of Fluid Imaging Technologies’ FlowCam Nano, released in August 2017, has improved the resolving capabilities of the technology, and now particles as small as 300 nm can be analyzed using flow imaging microscopy.

104 (4) 1440-1450 (2015).
	2. Gerhardt et al., 

103(6) 1601-1612 (2014).
	3. Tyagi et al., 

98 (1) 94-104 (2009).
	4. Maddux et al., 

General Chapter <788>, “Particulate Matter in Injections,” 

, p. 2722 (US Pharmacopeial Convention, Rockville, MD, 2006).
	6. USP, 

General Chapter <787>, “Subvisible Particulate Matter in Therapeutic Protein Injections,” 

, p. 547 (US Pharmacopeial Convention, Rockville, MD, 2014).
	7. USP, 

Informational Chapter <1787>, “Measurement of Subvisible Particulate Matter in Therapeutic Protein Injections,” 

, pp. 1680-1693 (US Pharmacopeial Convention, Rockville, MD, 2015). 


	Vol. 42, No. 6
	June 2018
	Pages: 34–36

	When referring to this article, please cite it as J. Markarian, "Identifying Contamination: Subvisible Particle Imaging," 

Flow imaging microscopy can be used to identify particulates and their sources.