Highly potent compounds can be defined as an active pharmaceutical ingredient (API) or intermediate with an occupational exposure limit (OEL) at or below 10 µg/m³ of air as an eight hour time-weighted average.¹ These compounds may have the ability to bind to specific receptors or inhibit specific enzymes and/or have the potential to cause cancer, mutations or other detrimental biological effects. Potency can be a function of structural class but it cannot be predicted for new molecules or new biological products without sufficient testing, therefore novel compounds of unknown potency and toxicity must be treated as highly potent to ensure safe handling in development.

For drug-substance manufacturing, the highly potent API (HPAPI) may be a small-molecule, biologic compound or antibody drug conjugate (ADC), which contains a cytotoxic small-molecule bound to a monoclonal antibody.

Largely due to continued high investment in oncology research and drug development, HPAPI is considered a high-growth area and currently comprises an estimated 25-30% of today’s candidate compound pipeline. The approximately 1,700 HPAPI in development today are utilizing parenteral, oral solid and other delivery routes to meet target product profiles.²

HPAPI, intermediates and finished dosage forms require specialized facility design, equipment, operation and process safety throughout the drug development process and commercial scale production in order to achieve the required level of containment and ensure operator safety. HPAPI is therefore increasingly being outsourced to specialized contract development and manufacturing organizations (CDMOs) with such infrastructure, expertise and track record in place. Particle engineering is often required to optimize API/HPAPI processing and performance, with specialized containment facilities—e.g., glove boxes—required to safely produce particle engineered drug product intermediates, e.g. micronized API.

HPAPI particle engineering via glove box technology
Activity of the compound (OEB vs OEL). The main aspect to consider when designing a glove box for particle size reduction—or micronization—is the Occupational Exposure Limit (OEL). The glove box must be designed to the lowest OEL value that will be handled in that specific unit to safely accommodate the range of HPAPIs that will be processed. Many sponsor companies specify the Occupational Exposure Band (OEB) value. While the OEL is a precise number, the OEB varies widely from company to company and cannot be used as common value. For example, whereas some sponsor companies have a four-level banding system, others may have a five- or even six-level system. As such, a requirement for an “OEB5 globe box” has no precise meeting. For adequate safety assurance, service providers must ensure that each band is fully covered with a specific containment solution and account for this inherent variation in OEB. An overview of the different OEB systems at sponsor companies is shown in Figure 2.

Compound properties and process considerations. Safe handling of a compound depends strongly on the specific process to be contained. The drier the product is, the dustier and more manual the process handled inside the glove box, and the higher the risk of inhalation exposure to the operator. In such cases, the glove box has to be designed with additional safety measures including additional pass boxes, liners, rapid transfer port (RTP) doors or laminar flows or a combination of these.

A glove box for the discharge of wet product from a centrifuge, or even a liquid from a reactor, does not require the same barriers against handling that micronizers or mills for dry powder commonly require.

Process design
Inlet/outlet set-up, processing volume and packaging methods. Once a glove box is closed, except in case of leakages, it’s safe. With negative pressure it is not possible for particles to exit. Leak tests and other evaluations (discussed later) are routinely performed to ensure that seals are fully tightened. Therefore, the weak point of glove box operation is during the transfer of material, parts or samples in and outside the glove box.

The design of the input/output (I/O) must be based on the number of samples, materials and quantities of active ingredient that will enter or exit the isolator. An optimized glove box design ensures flexibility, comfort and ease of operation, without compromising operator safety. A continuous liner or equivalent can provide a simple approach for safely introducing or removing any type of material, except those with sharp edges. Bags are universally accepted so their use does not limit subsequent processing steps. However, cutting and detaching bags is a fully manual operation dependent upon operator execution therefore additional protections (e.g. pass boxes, localize suctions, etc.) are required to guarantee containment.

Rapid transfer port (RTP) transfer systems lower human error, but these systems have limitations in terms of flexibility and capacity. They cannot be used to transfer large amounts of material and the glove box used for the adjacent steps of the process must be equipped with an Alfa-door, without which it is not possible to open the beta-container. Particle engineering service providers have to design in the flexibility required to accommodate the full range of packaging methods.

Airlocks and pass-boxes cannot be used as the only barrier from the main glove box chamber (dirty) because after some in/out operations these become dirty as well thus negating the protection they are intended to provide. This contamination is not necessarily apparent as nanogram and microgram sized particles are not visible. A combination of safeguards is therefore necessary, especially for reaching very high containment levels, to ensure a flexible and safe operation.

Mock-up studies
A mock-up study is the reproduction of the whole glove box (and sometimes this includes the surrounding room as well) in a 1:1 scale. During the mock-up study, the whole process has to be reproduced, from sampling to manufacturing and cleaning.

We consider this as the most important part of the manufacturing of a glove box and critical to conduct prior to installation and operation. Therefore, we make sure to dedicate enough time to mock-up studies which normally require two to three days.

Every detail of the glove box is tested and altered as necessary at this stage. Appropriately designed mock-up studies test for the expertise, patience and high manual skills required for the safe and efficient glove box operation. Parts that cannot be reached easily cannot be cleaned properly while parts too heavy are not ergonomic for ongoing operation. Titanium parts are used in such cases to facilitate efficient operation. Redundancy of safeguards that can offset operator error are also tested for during these studies.

HPAPI containment: SMEPAC testing
Glove boxes must be thoroughly tested on an ongoing basis to ensure proper functionality. Leak tests are necessary but do not provide full confidence. The leak test is a static test, executed when the glove box is not in operation. This should be executed daily to make sure that seals are fully tightened. However, passing the leak test does not guarantee the safety of the whole operation. As previously cited, the weak point of a glove box is when the active ingredient enters or exists the glove box and this risk must be fully tested out.

It is therefore necessary to run a “SMEPAC” (Standardized Measurement of Particulate Airborne Contamination) test, according to ISPE guidelines.³ The SMEPAC test consists in a series of pumps, with filtering membrane above them, placed around the glove box at points must likely to leak and on the operator. The pumps simulate the human breath, while the filters capture the particles during the simulation (using a placebo or a specific API) of the whole process, including inlet, outlet of material, the operation inside as well as the final washing and sampling.

The accuracy and precision of SMEPAC test results depend strongly on the material used (i.e., on the limit of quantitation [LOQ] of the analytical method used) and on the duration of the test (i.e., sampling time). The longer the sampling time, the lower will be the exposure limit that can be assessed. As an example, if a containment of 10 ng/m³ needs to be certified, and considering that the pump suction flow is fixed (e.g. 2L/min), the analytical method needs to have a LOQ to quantify up to 2 ng/m³. This means that the sampling time needs to be of sufficient duration to capture an amount of material on the pump membrane that after dilution result in a concentration up to the LOQ. To ensure that the testing is sufficiently representative, the SMEPAC test also needs to be executed under the same conditions as the final process (e.g., numbers of material inlet/outlet from the glove box, size and weight of the bags containing the material, sampling).

Particle engineering service providers are specialized in SMEPAC testing, analyzing the filters and calculating the operational exposure level while running on a specific glove box. We consider it important to run a SMEPAC test on a regular basis (six-month/annual intervals) to make sure the process is always safe for our operators.

Representative case study
According to the needs of a particular customer, the required containment level was determined to be 10 ng/m3 with the need for using multiple sizes of jet mills at standard and cryogenic conditions (-60°C). Moreover, a conditioning chamber attached to the milling section was required to reduce the amorphous content by reaching a defined temperature and relative humidity.

Layout of the glove box. The glove box design utilized was an “L” shaped unit, integrated into the cleanroom with the technical area behind the backwall of the unit. The right glove box is dedicated to milling and micronization, while the left one is dedicated to conditioning. They both share the same pass box, for both inlet and outlet. Each operation is run independently—the conditioning process can be run with the micronization chamber switched off.

The tightening is ensured by inflatable gaskets with active pressure monitoring and by an ISO class 2 validated glove box. Each window can be opened and is interlocked with the operation of the unit.

Inlet and outlet method. A flexible inlet and outlet solution is required and a double continuous liner solution utilized which allow for the removal of samples in bags. Due to the very low OEL (10 ng/m3) required with multiple inlet and outlet of both material and micronized HPAPI, we utilize a combined solution consisting of two liners and a middle pass box.

The design allows for micronized product to be collected into small glass bottles (primary packaging), and then moved into the pass box through a continuous liner (secondary packaging). The continuous liner (an ILC Dover system) is twisted, crimped and cut by the operator inside the pass box. Inside the pass box the operator wets all the surfaces with a wet cloth, to abate particles in the air and remove it from the outer bag. The bag is then moved into the conditioning chamber or outside (third layer of packaging), through another continuous liner, and also twisted, crimped and cut. Bags and material are introduced in the same manner, with the exception of the first usage when the chambers are clean.

Micronization. The milling/micronization chamber is predisposed for the assembly of three sizes of jet mills (MC50, MC100, MC150) for lab to pilot scale processing, a Hammer mill (HM100) and a pin mill (PM100). These mills all share the same volumetric feeder and cyclone filter. The environment is saturated with nitrogen to prevent dust explosions, with a relative humidity that can be increased up to 99% from the control panel. A scale is mounted inside for dispensing and feeding test purposes. The display, controls and the feeder’s motor are located outside the glove box so that only parts that can be washed with a direct jet of water/solvent are kept inside. The micronization line has an independent ventilation system, automatically controlled and with an in-line monitoring of airflow and temperature of the gas. The micronization can be performed at temperatures down to -60°C (-76°F) with the process gas cooled by liquid nitrogen, in an integrated heat exchanger, located in technical area below the conditioning chamber. The temperature is controlled by the PLC according to the flow of liquid nitrogen, fed by a movable Dewar located into the technical area behind the unit.

Conditioning. Fluid energy jet milling, depending on the level of grinding energy, can induce amorphous regions on the surface of the micronized HPAPI. Problems with respect to uncontrolled particle growth can arise when the recrystallization of the micronized material proceeds in an uncontrolled manner. A conditioning step following micronization is utilized to improve physicochemical stability of the material. During this process, the amorphous parts are converted into crystalline solids under storage conditions that are controlled with respect to relative humidity and temperature.
These conditions reduce the glass transition (Tg) of the HPAPI by the adsorption of water and setting the surrounding temperature to values above Tg so that the molecular mobility and, consequently, the recrystallization process is accelerated.

This approach makes possible the rapid and entire conversion of amorphous to crystalline material with minimum particle growth.

Since particular operating conditions, in terms of T, RH and storage time, need to be studied as a function of the physicochemical characteristics of the specific HPAPI, the whole chamber is designed to allow for a relative humidity up to RH 99% and a temperature up to 45 °C (113F).

Filtration and controls. Each glove box has an own ventilation and filtration system and works independently from the others. The reason for having different compartments is to ensure a complete air change of each portion for contamination, efficiency and safety reasons. In case of leakage from a glove or pressure drop due to broken glass, the accident is limited only to a portion of the unit, in order to prevent contamination and loss of product.

Each glove box has a double “push-push” filter on both inlet and outlet air, with an independent and separate line for the jet milling system. Each filter is monitored and can be replaced in a safe way from the technical area, without impacting the safety or the containment level.

Each parameter of the glove box, including temperature, humidity, scale, opening of the doors, washing procedures and milling/micronization, is controlled from the local Siemens Human Machine Interface (HMI), located on a movable arm next to the glove box. The HMI communicates with the distributed control system (DCS) and can be remotely controlled from it.

Each and every parameter is transferred to the DCS in order to prepare an automatic batch report. The software, CFR21 part 11 and GAMP5 certified, is tailored to the specific aspects of this unit.
Validation and SMEPAC test. The validation process includes FAT, SAT, IQ and OQ execution, with the protocol being tailored according to the requirements of the customer HPAPI. During these tests, all alarms have been executed and verified, together with the effectiveness of the micronization process and the functioning of all the utilities such as the temperature and humidity control system.

A series of pumps with the previously described procedure were located in the sensible points all around the glove box on the operator and at one meter from the unit. A total of three runs (as per ISPE guidelines) were carried out, with each of the runs executed with 19 pumps and the relative membranes continuously monitoring the environment and 5 SWAB. For two of the three runs, none of them reached or exceeded the measurable 2 ng/m3 against the requested 10 ng/m3 (i.e., 7 ng/m3 targeted). During the third run, only three points exceeded the 2 ng/m3, reaching respectively 6.2; 5.7 and 5.9 ng/m3. The geometric mean of the whole unit results in a containment level between 1.2 and 2.0 ng/m³.

Conclusions
Developing, manufacturing and engineering HPAPI requires much more than having processing equipment under containment. These compounds, which represent a growing percentage of the drug development pipeline, require specialized facilities and equipment design, operator expertise, and experience with process safety protocols. There are a number of approaches that can be utilized to ensure safe and effective particle engineering of HPAPI, and new technologies continue to be developed to enhance efficiency, ease of use and operator safety. Specialized service providers with an established track record offer an attractive and safe option for manufacturing and engineering HPAPI to meet drug target product profiles. 

References

1. A.W. Ader, J.J. Mason et al., Chem Today, 25(2), pp. 56-60 (2007).
2. Internal Lonza analysis, 2018.
3. ISPE. (2012). Assessing the Particulate Containment Performance of Pharmaceutical Equipment – Second Edition. Tampa, FL