Earlier this year the Commission on Genetic Modification (COGEM) commissioned Xendo to investigate the current vendor and user experiences concerning the containment risks when using Single-Use Bioreactors (SUBs) in combination with Genetically Modified Organisms (GMOs)
Based on this Xendo report COGEM has written a letter of advice to the state secretary of Infrastructure and Environment (I&M), concerning use of SUBs in large scale industry. In this letter, it is stated that production in SUBs by the current standard is performed under classification MI-III.
Following these events, we received questions regarding the report and the letter, so we’d like to summarize the outcome of the Xendo research report and present COGEM’s advice to the state secretary and how this should be interpreted. Also, we’ll show what Dutch regulations concerning genetically modified organisms state about MI classifications (Micro-organisms Industrial scale) with respect to the interpretation of the COGEM advice.
Summary Xendo Report: “Updated GMO Containment Risk Evaluation Of Single-Use Bioreactors”
SUB technology has developed significantly since the first assessment in 2010, with increased experience at both the user and vendor side, many improvements have been made that reduce
Since 2010 interaction between SUB vendors and end-users has resulted in increased knowledge concerning SUB bag integrity. One example is a visual anomaly database in which anomalies are classified according to their impact on bag integrity. SUB bag production processes are qualified and validated. Shipping and transport also occur according to validated methods. Training is provided extensively to users of SUB systems at purchase and when significant design changes have been made. SUB production has improved, yet between vendors, there still is a different approach to SUB bag integrity assurance.
Users often rely on the expertise and quality assurance of the vendor for bag integrity. Most users do not employ dedicated integrity tests (e.g. pressure decay), but instead use a media-fill-test as their pre-inoculation bag test. Users also recognize that maintaining a high level of operator skill, through (vendor) training and use of appropriate equipment, is critical to successful and safe use of SUB technology without containment events like spillage or leakage.
It is anticipated that small defects located in the headspace of SUB bags (pinholes) pose a (theoretical) risk for containment, especially when producing viruses which are transmissible through aerosols. Pinholes in the headspace are not easily found using a media fill test. A pressure decay test may not be able to indicate these holes as they have a minimum detectable defect size based on bag volume.
It is concluded that the use of SUB technology in combination with GMOs generally does not pose an increased containment risk compared to stainless steel vessels. This is based on the following:
- Any bag defect that could constitute a containment risk is typically spotted during the pre-inoculum phase. (Except the mentioned pinhole defects in the headspace)
- Operators are properly trained and use the appropriate and validated equipment.
- Vendors have good control over their production process and raw material supply.
- Vendors and users freely and openly communicate experience and information and engage in continuous improvement of this technology.
- Between different types of SUB bag configurations, no significant differences have been observed.
COGEM advice letter
COGEM presented the report to the state secretary of I&M and in the offer letter, COGEM explains the outcome of the Xendo research that has been done concerning the use of single-use bioreactors (SUBs) for
In the letter, COGEM indicates that accidental escape of GMOs through pinholes cannot be excluded and that operators should be made aware of the risks of pinhole defects. Nevertheless, COGEM indicates that, following the current classification regulations, large scale culture of GMOs in reactors and SUBs should be performed in
MI-III implies that besides the SUB itself there is a second level of physical containment. In MI-III classification the likelihood of GMOs being released into the environment is considered to be minimal, even when GMOs would escape from the SUB through accidental pinholes.
It should be noted that this classification advice has not changed with respect to the current classification of similar activities, indicating the Xendo report has confirmed the suitability of current classification guidelines.
Regulations concerning large scale production using GMOs
In the applicable Dutch regulations concerning genetically modified organisms (Besluit & Regeling genetisch gemodificeerde organismen, wet milieubeheer 2013) the following 4 classifications are listed for process installations for large scale industry:
- MI-I areas are for dealing with ML-I organisms that comply
tocertain criteria (very low risk). Inactivation of the biomass before disposal is not required.
- MI-II areas are meant for dealing with ML-I organisms that do not comply
tothe IAB criteria. Inactivation before disposal is required.
- MI-III areas are for ML-II organisms, and
- MI-IV areas are for ML-III organisms
Noticeably, when used at laboratory scale, all ML-I and ML-II organisms are classified at MI-III in industry. This is contradictory to classifications concerning PKM (Plants) and DM (Animal), where levels I to IV correspond to ML-I to –IV depending on the ML classification of the organism in laboratory scale work.
Appendix 5 chapter 5.7.1 states that:
- A. Dealing with organisms that are classified as ML-I in laboratory scale are classified as MI-III in industry.
- B. Dealing with organisms that are classified as ML-II in laboratory scale are classified as MI-III in industry.
- C. Dealing with organisms that are classified as ML-III in laboratory scale are classified as MI-IV in industry.
In general, the containment measures of MI classifications in the workspace entail the following:
- MI-I: Use of physical containment (Bioreactor).
- MI-II: Use of physical containment (Bioreactor) that limits the spread of GMOs. Filters in off-gas are mandatory.
- MI-III: Use of physical containment (Bioreactor) that strongly limits the spread of GMOs. Hydrophobic absolute filters in off-gas and means to collect and inactivate the total content of the system are mandatory.
- MI-IV: Use of physical containment (Bioreactor) that prevents the spread of GMOs. Further use of containment measures in the process area like a HEPA Filtered ventilation system, entrance through an anteroom containing a shower and gowning areas, a negative pressure regime, leak free or ventilated seals, hydrophobic absolute filters in off-gas and vacuum systems, and means to collect and inactivate the total content of the system are mandatory.
Also, procedural demands increase when the MI classification is higher. For further details see appendix 9 of the regulations concerning GMOs.
When applying for a license using ML-I or ML-II organisms at lab scale, always
The GMO office and COGEM have confirmed that their advice does not change the current classification rules and regulations. Downgrading is still possible when safety is assured.
Downgrade examples when using SUBs
Numerous applications for downscaling have been evaluated in the past. A number of positive COGEM
The outcome of the Xendo report is that use of SUBs overall does not pose a higher containment risk in comparison to stainless steel bioreactors. However, pinhole defects in the headspace of the bag are difficult to identify and therefore pose a (theoretical) risk of loss of containment, especially when using viruses that can be spread through aerosols.
COGEM has reported the outcome of the Xendo report to the state secretary of Infrastructure and Environment (I&M) indicating the risk of pin hole defects. However, COGEM states that under the standard MI-III classification risk is adequately managed because of the 2nd layer of containment besides the reactor itself.
The Dutch regulations concerning GMOs follow a standard classification of MI-III for industrial scale applications for organisms that on a laboratory scale are classified at ML-I or ML-II. It also states that a downgrade to MI-II or MI-I can be applied for at the GMO office.
The GMO office and COGEM have confirmed that, although the risk of pinhole defects in SUBs is acknowledged, there is no change in the classification of industrial application of SUBs. Furthermore, applications for downgrading to lower containment levels can still be submitted and will be evaluated on a case-by-case basis following current standards.