BPOG Response to Annex 2

Interpretation

It is proposed that decisions to assign area classification and implement other controls to prevent contamination should consider the nature of the operation, stage of the manufacturing process, and QRM outcome.

Clearly, the ingress of contaminants into process fluids or materials should be minimized to prevent adverse impact on the product. The risk-based approach requires development of justified control measures to potential sources of environmental contamination. This requires justification beyond simply assuming that filtration or other downstream purification steps remove environmental contaminants that enter during processing, or that contamination will be detected.

The following case illustrates the tenet that control measures should be commensurate with the risks (5) and the consequences of simple adherence to Annex 1 for biopharmaceutical processes:

One case where the risk profile of biological active substances manufacturing differs from sterile product manufacturing is an open aseptic process for inoculum preparation for cell culture. The operations often involve open manipulations of cell culture using aseptic processing techniques. Across the industry, Grade A conditions are achieved in the unidirectional air flow hood often used for such operations. The objective is to avoid microbial or viral contamination of the culture during these operations.

In the above example, adherence to all Annex 1 requirements for Grade A environments is not necessarily required to avoid contamination of the culture. However, since Annex 2 refers the reader to Annex 1 for principles and requirements of classified areas, one might conclude that an open operation, like inoculum preparation, where microbial contamination will result in failure of the batch, requires adherence to all Annex 1 background and in-process monitoring requirements for a Grade A environment.

Specifically, paragraph 33 from Annex 1 in the section “Aseptic Preparation” states:

“33. Handling and filling of aseptically prepared products should be done in a grade A environment with a grade B background.”

Hence, adherence to Annex 1 aseptic requirements would require a Grade B background environment for the Grade A environment for aseptic operations in biological active substances manufacturing. However, this interpretation may result in conditions that impede reliable manufacture of cell culture products. Application of Annex 1 requirements should be challenged and the design justified using QRM, to ensure the patient is being protected in the most appropriate way.

In the case of open operations that fill sterile medicinal products, Annex 1 applies and requires a Grade A environment with a Grade B background. This makes sense, as there are no additional steps to remove or inactivate microbial contaminants and limited opportunity to detect contamination before the product is given to the patient. In such a context, the Annex 1 requirement provides appropriate controls.

However, the Annex 1 requirements are less appropriate for inoculum preparation. Additional layers of protection are provided in the process and controls and mitigate the impacts of potential contamination:

• A microbial load or viral contamination would likely be detected through cell culture testing. Annex 2 acknowledges this point in paragraph 6 where it states “… contamination is likely to become evident during processes such as fermentation and cell culture.”

• Inoculum preparation is at the very beginning of many API manufacturing processes, with additional detection points and downstream process steps that remove contaminants.

Therefore, the risk profile of inoculum preparation differs from that of filling of sterile medicinal products. QRM evaluation will often indicate different requirements for this process step.

In current practice, industry has significant experience performing cell culture expansions in a Grade A hood with Grade C or Grade D background environments with very good success and exceedingly low contamination rates.

Regulatory authorities have supported this practice. For example, the Irish Medicines Board, Reference 6 states: “Typically, Grade C background with Grade A supply BSC is considered acceptable”. An Aide Mémoire (071210BN) issued by the German Health Authority (ZLG) indicates that a Grade A unidirectional flow hood in a Grade C background environment is appropriate for establishing a cell bank (7).

Interpretation

As stated in the previous section, paragraph 6 states that prevention of contamination is more appropriate than detection and removal. The authors agree with this approach so long as it is interpreted within a QRM framework. Some operations, such as solution preparation, are typically open operations, and microbial contamination is successfully removed by filtration. However, applying Grade C conditions to solution preparation in biological active substance manufacturing operations (as specified under paragraph 17 of Annex 1) may not be an appropriate control for a risk-based approach where many factors are considered.

In the following example for solution preparation, it is obvious that raw material is the primary contamination source as opposed to influences from the environment. Hence, controls developed to address bioburden from raw materials will also address bioburden from the environment.

Consider a solution preparation operation in which 1000 L of solution is prepared with a total solids concentration in the solution of 50 g/L. The solids used in the solution are manufactured under controlled, nonclassified conditions (at best). None of the solids are tested for viruses, but all are tested for bioburden and have a maximum bioburden specification of 100 CFU/g. This means that the final solution could contain 5,000,000 CFU of bioburden from the raw materials alone. This does not include microbial propagation in the solution during preparation.

The solids are added through an open port on the solution preparation vessel in an open manner. Environmental conditions should be set so that the potential additional contamination is negligible in comparison to the inherent microbial load in the starting materials. Even if the air contained 1000 CFU/m³, five times higher than the recommended values for Grade D specification in operation, 50 m³ of air (50,000 L) would have to enter the tank during the addition process in order to introduce 50,000 CFUs of bioburden, which is 1% of the maximum allowable amount of bioburden based on the raw material specifications. For a briefly exposed operation, exchange of 50 m³ of air between the vessel and surroundings is not credible, because both must be at the same pressure for the open operation. No driving force exists for rapid air exchange between the tank and the room environment. In this context, QRM should drive the appropriate manufacturing controls.

However, the tables in paragraph 17 and paragraph 32 of Annex 1 both mandate a Grade C environment for the preparation of solutions that are subsequently filtered when those solutions are used in a process in which the product is not terminally sterilized. This could lead to the interpretation that solution preparation for biological active substances, when the product is not terminally sterilized, should also be conducted in a Grade C environment. This should not be the case. Due to the inherently greater capability to detect and remove contaminants, the risk profile of biological active substance manufacturing is very different from manufacturing sterile medicinal products. Therefore, a risk-based approach should be taken when setting environmental conditions for such operations. See Appendix A for an example of the application of risk analysis to solution preparation operations. As a model, this example is intended to help when determining appropriate area classifications for such operations.

In summary, the two instances in which Annex 2 refers to Annex 1 may lead the reader to believe that Annex 1 also applies to the manufacture of biological active substance. Annex 1 is only referenced because it provides considerations and guidance for establishing classified environments that may be used to mitigate contamination risks if deemed necessary. In scenarios where Annex 2 is applicable, appropriate environmental controls should be implemented when the requirement is determined through the execution of a QRM process.

Interpretation

The degree of environmental control for contamination should be risk based and not driven by conventional practices or preset expectation. Elements to be considered when determining the risk of contamination from the environment are:

• Properties of the process materials, for example, growth promoting characteristics could affect hold time.

• The degree of closure of equipment.

For closed and functionally closed systems, the QRM process may determine that the risk of particulate or microbial contamination is minimal and that a CNC environment is appropriate, in which case the environmental monitoring program can be minimized or eliminated as required and appropriate for CNC environments. In the example below, the risk from room environment during cell expansion steps is rendered negligible by an approach to closing the operations.

A typical cell culture seed train process progresses through multiple cell expansion steps and increases batch volume in a process that requires multiple sterile transfers. In each transfer, connections are required that increase the process contamination risk by potentially exposing process contact surfaces to the outside environment. To minimize this risk it is critical to keep process vessel connections closed until ready to use and then to join them together while minimizing exposure to the outside environment.

The availability and successful use of sterile closed systems like presterilized disposable media bags and bioreactor bags that can be fitted with transfer, addition, and sample lines significantly reduce or eliminate the process contamination risks that otherwise can be a concern. The closed systems can be integrated through the use of techniques such as tube welding and/or disposable aseptic connectors, therefore ensuring that the process remains closed even when these connections are made. Because the entire seed train can now be performed as a closed system, it is not exposed to the room environment and the operation has been successfully performed without contamination in CNC areas.

The nature of the process step being executed can, of course, have an impact on how best to protect the drug substance, upstream versus downstream of viral clearance. The following example illustrates this for manufacture of a drug substance intermediate:

• Operations like fermentation are a classic example of an upstream drug substance manufacturing process. Fermentation processes are conducted in a closed system to prevent contamination (i.e., maintain pure culture) and QRM evaluation would support conducting such operations in CNC space.

• Downstream processes like purification of a drug substance have typically been located in classified areas. The higher room classification would often have been selected following a QRM evaluation, because purification operations typically have fewer layers of protection and often include open operations.

Implementation of QRM presents the opportunity to perform downstream operations in CNC or lower classification areas. Modern downstream processes often achieve a high degree of closure, such as utilizing functionally closed systems and single-use technologies (e.g., tube welding and sterile connections). With effectively closed systems coupled with other layers of protection, QRM evaluation may support locating the process in a CNC space, with minimal or no environmental monitoring requirements as determined by QRM evaluation.

For most systems, an effective way to minimize the risk of contamination is not to control the environment but to close the system so that the risk associated with the environment is minimized or eliminated.

Interpretation

Paragraph 50 focuses on the requirements appropriate for aseptic processing with no mention of the typical bulk biopharmaceutical nonaseptic requirements. The QRM process for entry into nonaseptic areas will indicate lower risk than for aseptic areas and minimal risk for entry into nonaseptic areas with functionally closed systems with no need for multiple wrappings and/or multiple stages of entry to the production area. Most of the discussion in paragraph 50 applies for the high-risk aseptic processes, but there is no discussion of lower risk options. Paragraph 50 could be interpreted as requiring multiple wrapping and/or stages of entry and surface sanitization requirements even for low contamination risk situations, such as for nonaseptic operations in closed systems. It is important to have the understanding that QRM evaluation and outcome should determine and inform the control strategy instead of incorporating process controls based on the nature of the process whether it is aseptic or nonaseptic.

Interpretation

It is essential to understand the probability or level of aerosol formation and spread and to evaluate the actual risk and consequences for cross-contamination posed by aerosols. Proportional containment measurements should be adopted according to the risk that has been determined. This may not require segregation or isolation of such operations when more robust solutions (like closed processing) are used.

This QRM approach is not limited to centrifuges and blending operations, the two examples mentioned in Annex 2. It should include any unit operation that can lead to aerosol formation.

Interpretation

Achieving viral safety of biological active substances requires more attention than simply undertaking pre- and post-virus reduction steps into separate rooms. Based on a sound QRM process and appropriate justification, use of physical barriers between pre- and post-virus operations (e.g., separate rooms or closed processing) is just one component of a complete risk mitigation strategy for virus contamination control. Other components include: barriers to virus entry (e.g., characterization of cell banks), raw materials sourcing, robust virus clearance steps, and testing during production to detect any contaminating virus (8).

Robust control strategies will only be achieved through a sound QRM process and not by solely following the current common practice. Where a virus inactivation or removal process is performed during manufacturing, the QRM approach should provide measures to identify and mitigate the risk of recontamination of treated products by nontreated products, components, or equipment.