	A number of factors can influence stability, especially with highly-complex biomolecules. The increased risk of instability requires more stringent practices to maintain stability throughout the shelf life of the product. In addition to this, measures need to be taken to minimize exposure to environmental factors that can affect the integrity and efficacy of a product.

	Kerry Bradford, analytical projects manager, and Ashleigh Wake, biopharmaceutical services leader, both at Intertek; Kim Cheung, senior director of quality at Genzyme; and Niall Dinwoodie, global coordinator of analytical testing, biologics testing solutions at Charles River, spoke with 

about the challenges in maintaining a stable environment for biologics, how to determine shelf life, the effects of upstream processing techniques on the end product, and biologics versus small-molecule drugs and the importance of stability testing.
	
	

: What standard methods are used to ensure cGMP requirements during stability testing?

: The standard test methods for stability testing are, on the whole, traditional quality control techniques for biologics. They include size exclusion; ion exchange and reversed phase high-performace liquid chromatography; sodium dodecyl sulfate polyacrylamide gel electrophoresis or capillary electrophoresis; potency assays; and physicochemical measurements such as appearance, pH, and particle size. Of these, particle-size measurements are gaining increasing focus, but the first test to fail is still often appearance.
	
	

: What are some of the challenges of maintaining a stable environment throughout testing?
	
	

: The main challenge here is the need for constant monitoring. Modern stability chambers are able to maintain conditions within the International Conference on Harmonization tolerances with little input, however, excursions caused by mechanical failure need to be quickly identified in order to prevent them from exceeding 24 hours. This requires a 24/7 alarm system to monitor all chambers and a dedicated team of staff to respond to emergencies and fix any breakdowns.

: The biggest challenge is controlling the condition of shipments, from stability chambers to testing locations and storage at these locations. Tracking chain of custody for stability samples, and maintaining sufficient back-up chambers and capacity ensures stability studies are not impacted by equipment malfunction or physical capacity constraints.

: Biologics present a number of challenges in ensuring a stable environment and comparability of results across time points. The glass transition point for highly concentrated protein products can occur within the normal operating range of some freezers. While this is relevant to real storage on the market, it can lead to a sudden change that has no reflection on the period of storage. Also, for frozen storage, consistency in thawing approaches are vital for data comparison between time points.

: What are some of the advantages associated with accelerated stability tests vs. real-time stability tests?

: According to the Arrhenius equation, samples undergo the same degradation after 32 days at 25 °C as after one year at 5 °C. There are obvious cost benefits to accelerated time periods, particularly to quickly eliminate poor candidates when at the early stages of development of either a new drug substance, drug product, or package. The accelerated tests can also be used to submit early data to regulatory authorities; however, accelerated storage data must always be backed up with real-time data in the long term.

: Accelerated tests can demonstrate comparability for material associated with process changes in a shorter period of time (e.g., six months) than long-term real-time studies. The tests can assess whether a degradation profile for an attribute is expected to be linear or non-linear. When temperature excursions happen during transport, there is a specific procedure that is followed to determine whether the product can be used or whether it must be discarded. The accelerated data are leveraged within that procedure in which we’ve documented the allowable excursion temperatures and times during processing, shipping, and handling, and the data are used to respond to physicians’/patients’ questions about mishandling.

: Accelerated tests are commonly used to confirm that the test methods being applied to the product indicate stability. The methods are qualified early on in a stability program to ensure that the real-time data reflect the true stability of the product.

: What are some of the challenges or disadvantages associated with using accelerated stability tests for the determination of kinetic degradation?

: While use of accelerated stability tests in conjunction with Arrhenius calculations is a good predictor of degradation rates for straightforward reaction kinetics, it does not account for physical changes in the sample, or more complex systems such as emulsions and suspensions. For example, elevated temperatures may promote slight solubility of a suspended drug particle, leading to significant degradation that would not occur in the equivalent room-temperature sample.

: For biologics, degradation observed under accelerated conditions is not necessarily predictive of long-term conditions.

: Complex molecules such as biologics rarely, if ever, obey the Arrhenius equation. There are different pathways for degradation to occur, both chemically and structurally, so no single degradation rate can be determined. The accelerated conditions will also influence the degradation observed and may lead to steps being missed or inappropriate molecules being targeted as the indication of degradation.

: What factors are involved in determining shelf life?

: Understanding the potential degradation pathways of any biologic is key to gaining a true shelf life. Achieving this understanding is definitely complicated by the structural diversity of biologics. Regardless, if we are working on an antibody, peptide, or oligonucleotide, our general approach is the same, and in many ways, weighted before any sample is placed on stability. During early stage development, the material will be stressed under extreme conditions of pH, temperature (60 ºC is high), light, and oxidative conditions, as well as physical forces such as prolonged agitation, orientation of storage, and freeze-thaw. The potential pathways of degradation are then evaluated using various analytical techniques such as circular dichroism (for higher order), Fourier-transform infrared, nuclear magnetic resonance, mass spectrometry, chromatography, and electrophoresis.

	The use of orthogonal approaches is, where possible, always applied in response to the complexity of analytics. The final stability program is then designed based on the results observed and the potential degradation pathways elucidated. Stability storage is then initiated accordingly, remembering that chemical/structural integrity is not enough. All biologic evaluations of potency upon storage should also be documented. A simple ‘box ticking’/checklist approach does not work for biologics, as what works for one will not necessarily work for another.

: Do upstream processing techniques have an effect on the stability of end products?

: The short answer is yes. However, this is very dependent on the drug and the downstream process. A lot of this is covered with assessment of the drug product as well as substance.

: For biologics, yes. For example, hold times of upstream materials can impact critical quality attributes and ultimately affect the stability of drug substances and drug products. Therefore, stability studies are executed on all intermediate production materials held longer than 24 hours.

 How does stability testing of biologics and small-molecule drugs compare?

 Issues associated with instability are potentially more significant and perhaps more likely to be observed with biologics than their small-molecule counterparts. Biologics are inherently unstable, and this can manifest in many ways, but what differentiates them the most is their tendency toward aggregation, a phenomenon which is both frequently observed and yet difficult to control. Aggregation can present considerable detrimental effects to the safety as well as efficacy of a molecule. Formulations are optimized to reduce or at least slow down the process of aggregation, but often, aggregation is the predominant change to the molecule observed on storage.

Both stability testing for large-molecule and small-molecule drugs are important to ensure that a product maintains specifications throughout its shelf-life. For small-molecules, critical quality attributes and degradation products may be better understood for each product and shelf-life specifications for the degradation products can be set based on toxicology studies. In my experience, there is more lot-to-lot consistency for stability testing of small-molecules, so interpretation of results is straight forward. Also, in my experience with biologics, stability testing encompasses many more product attributes and the degradation profiles may be inconsistent from product-to-product and/or lot-to-lot, making understanding identified trends and interpreting results more difficult. Test methods may be more variable, again making it difficult to interpret results.

 There is less predictability for biologics. Similar formulations of different proteins can behave in a different manner and have quite different shelf-lives, and the concentration often has a greater effect than it does for small-molecule drugs. The industry has seen many examples where process changes have led to changes in the stability of products that could not have been predicted. The use of committed stability tests for biologics is vital.

 When referring to this article, please cite it as A. Roberts, “Maintaining the Stability of Biologics,” 

Articles

Troubleshooting

Experts discuss crucial aspects of measuring the stability of biologics, which exhibit greater variability in stability testing than do small-molecule drugs.

Best Practices for Stability Testing of Biologics

	Twin-screw extrusion, a continuous mixing process, is gaining acceptance for producing solid-dosage drugs as developers deepen their understanding of how the process can be applied in pharmaceutical manufacturing. One commercial use today employs the well-established extrusion of plastic parts (e.g., fiber, tubing, or rods) and blends in a drug to produce a combination drug–device product. Another application, often called hot-melt extrusion (HME), uses a twin-screw extruder to combine excipients with APIs to improve bioavailability or for controlled release or taste masking. In addition, an emerging use of the twin-screw extruder is as a continuous granulator, which is an alternative to a batch granulator for agglomerating and homogenizing particles to improve powder-compaction properties for tablet pressing. Over the past decade, much has been learned about optimizing equipment set up and processing conditions for these applications, and research continues to develop further understanding and promote use.


	Although twin-screw extruders have been used in the plastics industry for decades and are well understood, using the equipment for granulation is relatively new. However, "a broad scientific community is investigating the specific requirements of pharmaceutical applications and their implementation with twin-screw granulation. So the level of understanding is growing quite rapidly," notes Dirk Leister, technical marketing leader, material characterization products, Thermo Fisher Scientific. A powerful aspect of the twin-screw extruder is its modular design; screw segments can be combined in various shapes and orders on the screw shaft to make a unique design that has a significant effect on product quality. The segments include conveying elements for moving material; mixing elements for shearing, mixing, or dispersing; and distributive flow elements for distribution of small quantities of additives or shear-sensitive APIs, explained Leister in a webinar (1). Although most research to date has been based on specific formulations, "There is already momentum around gaining a more universal understanding of how conveying, mixing, and distribution screw elements are influencing the granule formation and quality," noted Leister in an interview with 

	Some general screw designs have been determined for granulation. For example, mixing zones in granulation tend to be shorter and less intense compared to those used in HME, and a compaction section at the end of the screw (close to the discharge) should be avoided, says Leister. In addition to screw design, important parameters that affect the level of fines and porosity in granulation include feed rate, liquid/solid ratio, and screw speed. Barrel set-up and total length of the barrel, which is the reaction chamber of the granulator, also have an impact on the process, says Leister.

	An advantage of a twin-screw granulator is that it is easy to control, and parameters can be changed without stopping the process, which aids performing a designed experiment to define a process window. "Parameters like overall torque, temperature along the barrel, screw speed, and feed-rate can be monitored and adjusted quickly and precisely, and the twin-screw granulator will return to steady-state quickly," says Leister.

	"Industry and academia are aggressively tackling issues related to commercialization of this process," added Michael Thompson, professor in the Chemical Engineering Department at McMaster University in Canada, in a presentation at the Leistritz Pharmaceutical Extrusion Seminar (2). Researchers have come a long way in characterizing the mechanisms occurring inside the extruder. "Screw design is the single most important factor in affecting the granulation process," said Thompson, who showed how placement and pitch of kneading blocks and conveying elements, as well as wetting methods, affect the granules. Twin-screw granulation has matured so that it can be used effectively to eliminate downstream milling, but researchers have not been able to eliminate downstream drying, concluded Thompson.

	A benefit of twin-screw granulation that is driving its use is its flexibility, comments Leister. As a continuous, time-based (rather than volume-based) process, twin-screw granulation can process small or large volumes of material as needed. Although existing batch granulation equipment for validated processes and products is not likely to see wide-scale replacement at this point with twin-screw granulation, manufacturers are considering the continuous process for new products, concludes Leister, "We expect a significant growth in commercial twin-screw granulator projects within the next 3-5 years."

Evaluating screw design for hot-melt extrusion


	Hot-melt extrusion uses the same basic extruder equipment as twin-screw granulation but with a different screw setup. Several presenters at the Leistritz Pharmaceutical Extrusion Seminar described the influence of screw design (i.e., the type and placement of different screw segments on the shaft) on the mixing process and the properties of the end product.  

	Researchers at the University of Maryland (UMD), led by David Bigio, associate professor in the Mechanical Engineering Department, have developed a new tool to further evaluate the effect of twin-screw extruder design on product properties. Although many models use residence time distribution (RTD) as a way to characterize the effect of mixing, the group's experiments show that RTD does not directly correlate to the stress the material experiences. They have developed an approach to create a residence stress distribution (RSD) as a function of operating conditions. The approach employs polymeric beads that rupture at a specific stress. The percent break-up of beads quantifies the amount of material that experiences that stress and has been shown to be a function of screw speed and specific throughput. Masters student Ben Dryer presented his work in developing an equation for percent break-up and relating it to the final product properties of degradation, bioavailability, and residual moisture.

	"A single, three-dimensional equation that better predicts material behavior in the extruder could be powerful for computer modeling and improved understanding of twin-screw extrusion for pharma as well as other applications," concluded Bigio in an interview with 


	Researchers from Merck; the Research Center for Pharmaceutical Engineering in Graz, Austria; and simulation software company ANSYS presented work to model a HME process using one- and three-dimensional commercial modeling software (4). Improvements in such models enable software to be used to evaluate screw design and process conditions 

	Barriers to use of HME have been limited laboratory-scale equipment capability and the fact that not all APIs- depending on physicochemical properties such as melting point and organic solubility-are suited to HME. As a result, most early formulation work has been performed with spray drying, said Jim DiNunzio, head of the HME Technology Development Team at Merck, in a presentation (5). Recent modeling work and an improved process understanding are being used, however, to identify optimal processing conditions and move formulations developed in spray drying to HME. "We are now able to link characteristics assessed at small-scale to interactions in the melt state influenced by processing parameters, which facilitates using HME earlier in the development process. Eventually we hope to use HME earlier in development by utilizing small-scale predictive technologies supplemented with modeling and simulation to extrapolate at-scale performance," explained DiNunzio in an interview with 

	1.     D. Leister, "A Modular Approach to Continuous Granulation," webinar (June 24, 2015).
	2.     M. Thompson, "Progress in Continuous Granulation by Twin Screw Extrusion," presentation at the Leistritz Pharmaceutical Extrusion Seminar (Clinton, NJ, June 18, 2015).
	3.     B. Dryer, et al. "Pharmaceutical Application of the Residence Stress Distribution," presentation at the Leistritz Pharmaceutical Extrusion Seminar (Clinton, NJ, June 18, 2015).
	4.     D. Johnson, et al., "Hot Melt Extrusion Modeling," presentation at the Leistritz Pharmaceutical Extrusion Seminar (Clinton, NJ, June 18, 2015).
	5.     J. DiNunzio, "Formulation and Process Design of Melt Extruded Dispersions," presentation at the Leistritz Pharmaceutical Extrusion Seminar (Clinton, NJ, June 18, 2015).

Production Trends

Improved characterization of twin-screw extrusion enables increasing use in pharmaceutical manufacturing.

Twin-Screw Extrusion Makes Strides in Solid-Dosage Drug Production

	Stringent quality-control requirements, the need for more automated inspection, and an ever-stricter regulatory environment keep quality control top of mind for pharmaceutical packagers. As a result, many attendees visited this year’s INTERPHEX (held Apr. 21-23, 2015 at the Javits Center in New York) to see machines in action. In addition to optical inspection systems (see the 

), exhibitors gave attendees the opportunity to examine checkweighers, metal detectors, x-ray inspectors, leak detectors, and headspace analyzers in standalone or in-line systems.


	The HC-A checkweigher from OCS Checkweighers inspects products at 600 parts/min by measuring current in an electromagnetic coil at a rate of 1000 times/sec. A patented, dual weigh-cell configuration provides active vibration compensation and more accurate weight measurements. One cell captures product and all electronic noise, and the second cell captures only the noise so it can be subtracted, leaving a precise weight.

	Forced balance and anti-vibration technology on the Ishida DACS-6 checkweigher from Heat and Control ensure extremely accurate weighing. The sanitary design is washdown-compatible and features tool-less belt removal. Integration with a metal detector results in a dual-purpose system.

	Another dual-purpose system, the Ensura model from ACG North America, integrates checkweighing with serialization and vision inspection. The compact machine adjusts for different carton sizes and accommodates virtually any type of printer. Variable speed control enhances flexibility.


	Upgraded software adds functionality to the Ceia THS/PH2IN metal detector from Heat and Control. New features include networkability; automatic maintenance alerts; capture, storage, and analysis of performance data; and timed-interval email reports. The all-digital machine eliminates the need for physical calibration. Wi-fi, ethernet, and USB connectivity enables monitoring and adjustment via smartphone.

	Insight metal detectors from Loma Systems check tablets and bottles. Generally installed after the tablet press, the smaller Insight Pharma Unit may be specified with a 95 x 38-mm or 95 x 22-mm aperture. Detecting sensitivity ranges from 0.3 mm for ferrous to 0.35 mm for nonferrous and 0.5 mm for stainless-steel contaminants.

	The PSG Now metal detector from PPi Technologies Group also checks tablets and is compatible with capsules.


	X-ray inspection systems sometimes perform the same role as metal detectors, but also can check for other types of contaminants as well as product features. For example, the Xavis x-ray automatic vision inspection system from PPI Technologies Group also locates broken product and incomplete fills, and it confirms seal integrity on pouches and blisters. 

	The Ishida IX-GA-S-2462 x-ray inspection system locates foreign, broken, and missing product and also provides weights. An auto-learn feature simplifies setup via use of a known good sample. Sanitary design includes a one-piece, removable, washdown-compatible conveyor.

	The G180 x-ray system from Sapphire Technologies USA can be configured to check trays, pouches, cases, bottles, cans, inhalers, pumps, and loose product. High resolution helps the unit locate contaminants; confirm count, mass, and fill level; and detect broken, damaged, or incomplete products.


	Lasers in the Vista in-line headspace inspection system from Lighthouse Instruments monitor oxygen, pressure, and moisture levels to confirm container/closure integrity, oxygen levels during filling, and moisture levels in lyophilized product in vials from 1 to 200 mL. Rated at up to 200 vials/min, the system also can detect raised stoppers. In addition, since microbes typically consume oxygen and release carbon dioxide, the unit can identify microbial contamination in media fills by detecting changes in headspace oxygen and carbon dioxide levels. Compared to standard incubation tests, which take 14 days, the laser-based system can deliver results in as little as 24 hours.

	The dual-cycle Veripac 455 leak detector from PTI–Packaging Technologies & Inspection looks for large leaks then checks for smaller ones. This sequence is used because if a large leak is present, gas would escape, rendering smaller defects undetectable.

	Some leak detectors check container integrity with high voltage (HVLD). Seidenader Maschinenbau has added the standalone HVLD Pro system to its integrated high-voltage leak detector/camera system. Replacing timing-screw transport with servo-motor control reduces variation in the distance between electrode and product and ensures a 360° rotation of each container. An adjustable vial angle maximizes the range of products that can be tested.

	The HAS-Lab headspace analyzer from Wilco USA now measures carbon dioxide as well as moisture and oxygen. Carbon dioxide measurements are useful for media-fill analysis and cold-chain process problems. “It’s important to be able to prove elastomeric components won’t be affected by exposure to cold temperatures and leak,” Paul Bilotti of Wilco told 

. The nondestructive, laser-based testing system relies on up to three lasers: one for oxygen (760 nm), one for moisture (1340 nm), and one for carbon dioxide (about 2000 nm), with the most common units pairing oxygen and humidity. 

	Mass-extraction technology, which detects leaks as small as 2 microns on a 10-mL vial, is the basis for the ME inspection systems from ATC. Vials enter a vacuum chamber, where a patented flow-measurement sensor detects any outflow of gas from the container of liquid or lyophilized product. The system also can check prefilled syringes and intravenous bags. Models include the standalone ME2 and the fully automated, multi-station ME3.

Packaging

Checkweighers, metal detectors, x-ray inspectors, leak detectors, headspace analyzers, and optical inspection systems for packaging were demonstrated at INTERPHEX 2015.

Equipment and Processing Report-07-15-2015

Best Practices for Stability Testing of Biologics

Twin-Screw Extrusion Makes Strides in Solid-Dosage Drug Production

Quality Control Solutions at INTERPHEX: Inspection and Detection Systems