1.0 Introduction
Antibody-drug Conjugates or ADCs are a new generation of highly hazardous / highly toxic pharmaceutical products used, among other things, in the targeted treatment of cancer. Most of these ADCs require an Occupational Exposure Limit or OEL below 50 nanograms/m3.
The manufacture of Antibody-drug Conjugates or ADCs offers a new challenge, particularly in aseptic production. In order to meet Current Good Manufacturing Practice or cGMP requirements the aseptic process must be run in positive pressure, and the operating personnel must also be protected actively from the substance. Isolators have been used successfully for many years for product protection in aseptic manufacture, and they are now being called on to provide active personal protection as well. Is this a contradiction in terms? At first glance the answer is yes. In aseptic manufacture, isolators are operated in positive pressure in order to protect the product. Containment, however, calls for negative pressure in the isolator to protect personnel by preventing the hazardous substance from escaping. Special seals on the isolator and a sophisticated pressure-cascade concept with active mouseholes make it possible to protect both the product and personnel.
Introduction: Where do these Occupational Exposure Limits or OELs come from?
2.0 How are OELs calculated?
Occupational Exposure Limits or OELs are calculated on the basis of Acceptable Daily Exposure / Permitted Daily Exposure or ADE/PDE. ADE is used specifically in the USA, while PDE is a European term from the EMA (European Medicines Agency) that has been mandatory in the manufacture of pharmaceutical products since 2015, as outlined in the EU GMP Guideline, Chapter 5, Article “Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities“.
One of the sections of this guideline is entitled ‘Data requirements for hazard identification.’
| Worker ADE/PDE in mg/day = | NOEL (mg/day) UFc x BA |
NOEL = No Observed Effect Level
UFc = Cumulative Uncertainty Factor for the worker
BA = Bioavailability by inhalation
ADE = Acceptable Daily Exposure in mg/day
PDE = Permitted Daily Exposure in mg/day
The relationship between the ADE/PDE and the OEL can also be seen in the Containment Pyramid (Figure 1.0). Alongside the OEL, the ADE/PDE is also used to calculate threshold values for cleaning residues and for product carry-over from one substance to the next. The threshold values calculated using PDE also replace the 10 ppm and the 1/1000 of the daily dose criteria previously applied in the European Union.
3.0 Adequate containment for complying with the required threshold value of 50 nanograms/m3
What do we mean when we refer to a threshold value of 50 nanograms/m3? Let us take a particle with a size of 0.5 μm. This particle size is relevant in the classification of clean rooms. Cleanroom class A (ISO class 5) allows for 3,520 particles (>= 0.5 μm) per m3. The weight of a 0.5 μm particle with a product bulk density of 0.8 kg/l is approximately 100 nanograms. With a required OEL of 50 nanograms/m3, therefore the particle is said to have a size of 250 nanometers or 0.25 μm. A series of important measures must be taken in order to achieve control over this 250 nanometer- particle with regard to cleaning, and to complying with the OEL.
4.0 Measures for achieving a threshold value of 50 nanograms/m3
The critical stages in the manufacturing process of ADCs are producing the toxin ‘payload’ – a highly active substance that requires a defined OEL – and its linker, and adding the payload to the conjugate. Following sterile filtration, filling the product into vials and freeze-drying are further critical stages.
Which technologies exist for meeting the requirements of extremely low OELs?
In the last decade, a whole host of technologies have been developed with a view to transferring highly active or highly hazardous products safely into or out of a manufacturing process. When it comes to safely remaining below an OEL of 50 nanograms/m3, however, isolator technology is used in most cases. Originally developed for the nuclear industry, isolators have also been in use for many years in pharmaceutical manufacturing for highly active or highly hazardous substances. Isolator technology involves the use of a contained space, to which access is obtained by means of gloves attached to a glass door (Photo 4.0: ADC aseptic fill and finish). The interior of the isolator is operated in negative or positive pressure, depending on the requirement. For straightforward personal protection, the isolator is operated in negative pressure in order to prevent the hazardous substance from escaping. In aseptic manufacture, product protection has top priority and therefore requires the isolator to be operated in positive pressure.
Prior to the sterile filtration of the ADC, personal protection has priority, which is why straightforward personal protection isolators operated in negative pressure are used up until this stage. Isolators also vary in ways that can make them suitable for substances below 50 nanograms/m3.
The following requirements apply to an OEL of 50 nanograms/m3.
- A suitable airlock system for inserting the highly active substance into the isolator, as well as for removing material and waste
- High-performance filter technology
- Hygiene design for cleaning on product changeover
- Glove testing
- Transfer of payload into conjugate container
5.0 Airlock systems
The most common design mistakes occur with the airlock system into and out of the isolator, the filter technology, the gloves and the transfer of the substance into the conjugate vessel.
The airlock system is needed to insert the highly hazardous pharmaceutical substance into the isolator, as well as to remove empty containers or product residues from the isolator. There are various systems available for carrying out this transfer.
Possible airlock systems:
- Transfer airlocks with locked doors between the pre-chamber and the main chamber of the isolator
- Endless liner technologies
- Rapid transfer ports (RTPs)
These systems all have weak points of varying significance that could cause a possible containment breach. Furthermore, these systems often fail to meet OELs below 50 nanograms/m3. The form of containment necessary to achieve an OEL of 50 nanograms/m3 comprises a combination of two different barrier systems – e.g. a Rapid Transfer Port (RTP) attached to an airlock on the isolator, and a locked door from the airlock into the isolator, with the pressure remaining higher in the airlock than in the main chamber of the isolator. Another dual barrier system is an airlock with an endless liner system between the material airlock and the main chamber.
6.0 Filter technology
The filter technology used together with the isolator is another critical area. Given that personal protection isolators are operated in negative pressure, suitable filter technologies are also required at the air inlet and outlet points of the isolator.
Possible filter systems:
- Bag-in/bag-out filter
- Push-push filter cartridge
- Filter cartridge (FiPa)
All of these filter systems are suitable for preventing highly hazardous substances from escaping from the isolator. Some filter systems present a GMP risk, however, as bag-in/bag-out filters and push-push filter cartridges can cause recontamination of the new filter by the contaminated one during filter change, for example. Particles can then become detached from the new filter and enter the area containing the next product, and this is critical because this recontamination is often not discovered. The filter cartridge (FiPa) avoids this.
FiPa filter technology was developed as a filter isolator. The FiPa is designed as a closed system that is attached to the isolator, with the opening on the FiPa towards the interior of the isolator operated from the outside. The dust-laden air can enter the FiPa, and the dust is deposited on the filter. During product changeover, the FiPa is sealed again from the outside. The isolator can now be cleaned, and after cleaning the FiPa is removed with no risk to the operator or to the next product to be manufactured.
In their paper “Safe Change Filter Systems for Containments in the Pharmaceutical Industry,” published in Die Pharmazeutische Industrie (September 2011), Frank Lehmann and Jörg Lümkemann explored the various issues of contamination-free filter change. [1]
Hygiene design and glove testing
The concept of hygiene design refers to the cleanability of the interior surfaces of the isolator or of devices built into the isolator. As mentioned above, cleanability is hugely important as it represents the greatest risk of cross-contamination between two consecutively manufactured substances from a GMP perspective. Particular attention must therefore be paid to the seals on the front glass panels when it comes to designing an isolator. Static seals are often used and thus also the most problematic because they wear out over time and can allow dust to be deposited and penetrate critical areas. This process of wear can also affect the airtightness of the isolator. Following cleaning in particular, opening the glass panel can cause the product residues deposited in the seals to become detached and escape from the isolator. Inflatable seals are a better option in terms of hygienic design. With an optimal design, these seals allow for highly accurate sealing of the glass panel, and the seal and its functionality can be tested using validated measuring equipment.
Another important point is the design of the seals for gloves, as well as glove testing. When it comes to attaching the gloves there are also various possibilities for ensuring that the necessary level of containment is achieved. Most of these options make use of a double o-ring groove, which is needed to ensure a closed changeover in the event of a damaged glove.
From a containment perspective these o-ring attachments are a weak point, as containment cannot be achieved if the gloves are fixed incorrectly. It is also important to prevent the highly active or highly hazardous substance from accessing and becoming deposited around the o-rings, as this area is critical when it comes to cleaning. An ideal solution is an additional seal on the o-ring and on the sleeve of the glove, in order to prevent the substance from reaching the o-ring.
Glove testing is carried out to complete the safety check of this area, and involves examining the gloves for small tears or pinholes. The gloves are used to work inside the isolator, and are therefore exposed to risk of damage. This damage inspection should be carried out on a regular basis.
In their paper “How Risky are Pinholes in Gloves? A Rational Appeal for the Integrity of Gloves for Isolators,” published in PDA Journal of Pharmaceutical Science and Technology (2011), Angela Gessler, Alexander Staerk, Volker Sigwarth, and Claude Moirandat, Ph.D, describe different glove integrity test procedures and their ability to detect leaking gloves as well as results from extensive microbiological tests performed to give more evidence and crosscorrelation to physical testing.[2]
8.0 Transfer to the conjugate vessel
If the toxin/payload is in powder form, transfer to the next process via a powder transfer system should be avoided. The transfer systems currently available are not completely adequate for this step, and it is more advisable to mix the powdered substance with a suitable liquid within the isolator. Once the highly hazardous substance has been dissolved in liquid, it should be transferred to the conjugate container either via a peristaltic (tube) pump or via a “AT Connect” connector.
Both of these options involve single-use systems that facilitate safe transfer. With the peristaltic pump it is important to ensure that the product hoses connecting the conjugate container are cleaned before being removed. Here it is important that the hose connectors are designed in such a way that spillage is prevented when the hoses are disconnected. The “AT Connect” system makes this possible thanks to a special connecting and disconnecting process, and was developed to transfer sterile liquids from a container into an isolator for sterile filling into vials or syringes.
The same principle can also be applied to the closed transfer of a liquid from an isolator to the conjugate vessel. The passive adapter of the “AT Connect” system is attached to the conjugate vessel. The active part of the “AT Connect” adapter is located in the isolator, and the passive part is connected from the conjugate vessel to the active part of the isolator and locked in place. Once it has been locked, the active part can be opened and connected to the liquid container in the isolator, to allow for safe, closed transfer.
9.0 Aseptic fill and finish
Once the ADC has been sterile filtered, the fill and finish process can take place. It consists of the following critical steps:
- ADC transfer to the vial-filling area
- Vial-filling
- Freeze-drying (lyophilization)
- Inspection
- Packaging
The entire manufacturing process from vial-filling to the freeze-dried pharmaceutical product takes place under cleanroom class ISO 5 conditions. Given that the ADC product is a highly hazardous substance, it is also recommended that these manufacturing steps be handled using isolator technology. Isolator technologies are widely used in aseptic manufacture, and have the benefit that operating personnel have no direct access to critical aseptic areas.
Based on the Fractional Negative method of determining the D-values of Biological Indicators (BIs), contained in the ISO 11138-1 and EN 866-3 standards, Sigwarth and Moirandat describe the requirements regarding aseptic manufacture in isolators and discuss a complete and systematic method that enables the parameters for each cycle phase to be determined and verified as well as the effectiveness of the process to be quantified. Their method also enables differences in bacterial reduction between positions which can be effectively decontaminated and “worst case” positions to be quantified. Sigwarth and Moirandat further describe how these quantified results can be used to individually adjust a process to specific overall bacterial reduction requirements. [3]
In another paper, Patrick Vanhecke together with his colleagues Sigwarth and Moirandat present major criteria for the effective use of fumigation with emphasis on a new H2O2 procedure which focuses on a proper and simplified validation of a process using standardized biological indicators with defined concentrations. The new concept for validation of the sanitization procedure overcomes the problems associated with conventional surface disinfection validation. It allows for considerable more safety at greatly reduced cost and work.[4]
In accordance with GMP guidelines on aseptic manufacture, isolators are operated in positive pressure in order to protect the product. When it comes to protecting personnel, however, the isolators should be in negative pressure in order to prevent the active substance from escaping. But how can these GMP and personal protection requirements fit together?
This double safety level can be achieved by incorporating additional safety systems. These safety systems are essentially similar to those used in the personal protection isolators described in this article, namely special seals on the glass panels and regular inspection of these seals, FiPa technology to prevent the highly hazardous substance from reaching the air circulation channels and an absolute hygiene designed Isolator.
It is also necessary to prevent the substance from being spread through the isolator should a vial happen to break, and this is achieved by means of various pressure cascades. The more critical the area, the lower the pressure to the other areas. Targeted air flows to the FiPa filters also reduce the spread of the substance, particularly during vial-filling and the unloading of the freeze-dryer. These measures enable OELs below 10 nanograms/m3 to be achieved, as verified on the basis of the ISPE Good Practice Guide “Assessing the Particulate Containment Performance of Pharmaceutical Equipment.” [5]
Aiming to define current good practices, the second edition of the ISPE Good Practice Guide provides information designed aid organizations in benchmarking their practices and improve on them. The guide has been updated to address a broader selection of containment technologies and processing equipment and provides technical guidance and consistent methodologies for evaluating the particulate containment performance (particulate emissions) of pharmaceutical equipment and systems.[5]
10.0 Packaging
Following the freeze-drying process, the dried powder is contained in sealed vials. These vials are washed before they leave the isolator in order to prevent any contamination. The risk of a vial being broken during inspection and packaging remains, however.
The inspection and packaging of the vials must for this reason also be protected using suitable isolator technology in the critical areas.
11.0 Summary
ADCs are a new generation of highly active and extremely hazardous substances in the pharmaceutical industry that call for a new kind of containment for their manufacture. While the quantities manufactured in the initial phase of development are low, there is a significant risk of coming into contact with the product if adequate protective measures are not taken. Isolator technologies are suitable for this purpose, but require innovative solutions and safety precautions when it comes to handling ADCs safely.
April 4, 2016 | Corresponding Author Richard Denk | doi: 10.14229/jadc.2016.04.02.001
Received: February 28, 2016 (German) / March 20, 2016 (English) | Published online April 4, 2016 | This article has been peer reviewed by an independent editorial review board.
Disclosures: Richard Denk is Head Sales Containment at SKAN AG and ISPE chair of the COP Containment DACH.
