Practical Considerations for Demonstrating Drug Substance Uniformity

ABSTRACT

Drug-substance uniformity is an important consideration for the final step in the manufacture of drug substance/active pharmaceutical ingredient. Uniformity studies are necessary to ensure that the entire contents of the batch are homogenous and that the drug substance specification sample is representative of the batch. This paper describes considerations for drug-substance uniformity, such as selection of appropriate test parameters and sample points, and approaches to establishing acceptance criteria. Additionally, operational considerations and best practices to ensure robust and consistent drug substance filtration and uniformity are described.

The final step in drug-substance (active pharmaceutical ingredient, API) manufacture is typically a 0.2 µm filtration step. This filtration step serves as a final clarification of the process pool and also as a bioburden control measure prior to the storage and further processing of drug substance (DS) to drug product (DP). There are many technical considerations to ensure a consistent and robust bulk filtration step, for example:

• Selection and sizing of the filter

• Materials of construction for the DS containers and tubing for transfers

• Filtration equipment procedures

• Filter preparation to reduce extractable and leachable components

• Container–closure integrity.

One aspect that must not be overlooked is the DS uniformity (or homogeneity) which is discussed in this article. When designing validation studies, it is important to consider the stringent regulatory expectations for ensuring batch uniformity and integrity of drug products (1).

The DS may be filtered into single or multiple vessels. In the former case (single agitated vessel), uniformity considerations are mixing speed and mixing time prior to taking a sample that is representative of the entire contents of the vessel. In the latter case, it is necessary to demonstrate uniformity between the DS containers. The initial concentration of product effluent from the filter may be expected to be slightly lower than the rest of the pool due to dilution with residual flush solutions in the filter pores and housing assembly, as well as due to non-specific protein or excipient adsorption to the filter. Thus, uniformity acceptance criteria should take into consideration an asymptotic increase in concentration during a reasonable initial product volume. This paper describes risk-based approaches to establish rigorous acceptance criteria and define operational parameters to ensure consistent DS uniformity.

RELEVANT PERFORMANCE PARAMETERS FOR A DRUG-SUBSTANCE UNIFORMITY STUDY

The DS specification parameters for biological products confirm the identity, purity, potency, quality and safety of the API. The ultimate aim of a DS uniformity study is to ensure that individual DS containers are consistent with respect to all of these critical quality attributes (CQAs) or specification parameters. For the purpose of demonstrating uniformity, it is not necessary to test each of the DS specification parameters, since a subset of the parameters may be used as a surrogate for the others. Typically, the quantitative specification parameters can be used to demonstrate uniformity. Representative assays may include protein concentration by absorbance (e.g., UV280), high performance liquid chromatography (HPLC) or bioassay. Of these, the UV280 is the simplest and fastest measurement with an acceptable degree of accuracy and precision. Protein concentration also allows evaluation of possible mechanisms for introduction of non-uniformity to the DS batch (e.g., dilution due to residual flush liquid, protein adsorption to the filter material, or inherent uniformity challenges with the upstream pool). Other parameters, such as pH, osmolality, conductivity and/or purity (e.g., aggregate concentration) may also be used as measures for DS uniformity. It is important to consider the potential failure modes for uniformity when selecting performance parameters such that they are sensitive enough to pick up a lack of uniformity. For example, if dilution with residual water is a potential source for non-uniformity, pH may not be the best parameter to use because water for injection (WFI) may not significantly impact the buffering capacity of the drug substance formulation buffer. Additionally, stabilizing agents such as polysorbate may be a critical component of the DS and it may be necessary to demonstrate that the concentration of such agents is within required limits. Other factors, such as shear could impact DS product quality (particle size, aggregation, potency), and a risk assessment should be considered to identify and mitigate these potential outcomes. Validation provides evidence of sufficient uniformity such that any sample location across the fill is representative of the entire DS lot with respect to the CQAs. Validation allows batch release and stability testing to be performed on a representative sample collected from a single location within the DS fill operation.

Figure 1: Flow diagram of drug substance filtration. (FIGURE IS COURTESY OF THE AUTHOR)

Another key question is when to take the samples to demonstrate uniformity and how to establish appropriate validation acceptance criteria. Typically, samples may be taken at the beginning, middle, and end of bulk filtration, and if they meet prospective acceptance criteria, the entire batch can be considered to be homogenous. The sample collection strategy is an important consideration: Does one take a point sample (i.e. directly from the filter bell), or a pool sample from the actual container? The latter is more relevant but needs to be balanced against the potential risk of contamination during sampling, and mixing considerations prior to taking the sample. Typically, the beginning sample is taken as a pool sample from the first container, as this reflects how the contents of the DS will be forward processed. If the DS lot-release and stability samples are taken as point samples at the middle of bulk filtration, the use of point samples during a uniformity study may be favorable for consistency and to minimize potential for contamination.

There are several ways to establish acceptance criteria to demonstrate DS uniformity, which may be used in isolation or in combination, as appropriate. Prior to selecting a preferred strategy, it is important to understand how the DS will be used during the subsequent DP formulation and testing, and to ensure that the requirements for these steps are addressed in the DS approach. Several methods for DS testing, and their pros and cons, are briefly discussed below.

Calculation based on DP specification limits

This method is based on a back calculation of the requirements for DP processing and DP specification criteria. The DS batch may be divided and used over several DP batches. In order to maintain this flexibility, it is necessary to ensure that any DS batch or part thereof used for DP processing will meet the DP specification requirements. It is necessary to take into account the smallest DP stock keeping unit (SKU), including product concentration and manufacturing volume, which will be used. The advantage of this method is that it is based on processing needs. However, it may be complicated by the existence of multiple SKUs and the desired safety margin between a passing uniformity result for DS compared to the associated specification range for DP.

Requirements at or tighter than the DS specification limits

The criteria for DS uniformity may be established to be the same as the DS specification limits. While this is a relatively simple way of setting the uniformity process validation acceptance criteria (PVAC), it does carry the undesirable risk of failure for DP specification and uniformity limits if the sample measurements are at the DS specification limits, especially if there are stability concerns during storage. One way to reduce this risk is to set the validation acceptance criteria within the DS specification limits, thereby providing a safety margin. One should also ensure that the documented analytical method precision is able to achieve results of the reduced range. The advantage of this approach is its simplicity in applying a safety margin relative to the specification limits, which ensures robustness in meeting the process needs. The downside is that the safety margin allowance may be subjective and may not align with the method precision.

Table I: Typical processing steps in drug substance filtration operation. (FIGURE IS COURTESY OF THE AUTHOR)

Inclusion of analytical method variance (to account for potential measurement error)

This strategy takes into consideration all potential errors that could contribute variability to the uniformity results, such as analytical method variability and volume measurement for the product and additives, in order to ensure acceptable DS uniformity. The root mean squares of the potential errors are calculated to derive the acceptance criteria. The acceptance criteria may be established based on the analytical method variability or precision when the analytical method variance is significantly greater (more than one order of magnitude) than the process variance. It should be emphasized that the prospective method validation acceptance criteria should be used in the calculation, rather than the results from the analytical method validation exercise. One must also ensure that sound technical justification exists for how the method validation acceptance criteria are developed. The latter is typically based on a fairly small data set and could result in overly tight acceptance criteria, resulting in a failure of the uniformity study. The acceptance criteria is set so that the percentage relative standard deviation (RSD) of the sample points is less than or equal to the analytical method precision. An assessment on process impact in using this method is recommended to ensure that validation acceptance criteria are not set too wide.

Percentage of the feed stream concentration

Another simple way to establish uniformity study validation acceptance criteria is to evaluate individual samples against a percentage of the feed material concentration. Selection of the actual percentage value could use a similar approach to that described the two previous methods. This method is based on the assumption that typically, there may be a small amount of product adsorption onto the bulk filter or dilution from flush water/buffer retained in the filter apparatus. As such, this source of variability is expected during the initial phase of the bulk filtration step. This method is applicable where the principal uniformity failure mode is based on dilution. It is fairly simple to implement, but can be problematic if the method variability is high.

Figure 2: Concentration curves for surrogate (0.1 M NaCl) and product filtrations. (FIGURE IS COURTESY OF THE AUTHOR)

Tolerance interval

This approach may be used if adequate historical filtration data exists to calculate a tolerance interval and capture the expected long-term behavior of the DS fill process. Typically, the tolerance interval contains 99% of the population (coverage) with a 95% confidence limit. However, the confidence and population coverage depends on the size of the historical data set. Care should be taken in combining data from laboratory or pilot scale filtrations with full–scale or manufacturing data, as the non-recoverable volume of the systems may result in substantial differences in terms of batch uniformity. Additionally, the calculated uniformity limits should not be wider than the DS/DP specification limits. While this method is based on actual historical experience, it does require additional sampling and testing during clinical lots.

Equivalency acceptance criteria

The objective of the equivalency approach is to test the null hypothesis of non-equivalence (non-uniformity) within a DS batch. If the null hypothesis is rejected, evidence of uniformity within a batch is demonstrated. Multiple samples are required for each sample point within the batch, and the means are calculated for each sample location. Uniformity within a batch is demonstrated when proscribed confidence intervals (typically at 90% or 95%) of the difference between the means are within the calculated acceptance criteria. The benefit of this equivalence acceptance criteria (EAC) approach is a statistically defined proof of uniformity within the allowable variability. However, this method does require a larger number of samples to be collected from each sample point in order to have sufficient statistical power to make the acceptance criteria meaningful.

–EAC<µ1 – µ2<EAC

–EAC<µ1 – µ3<EAC

–EAC<µ2 – µ3<EAC

where:

µ1, µ2, µ3 are the mean of the sample test parameter over the course of bulk filtration (e.g. protein concentration at the beginning, middle and end), and EAC is the equivalency acceptance criteria. It is beyond the scope of this paper to describe equivalence testing; References 2 and 3 provide general sources on statistical equivalence.

OPERATIONAL PARAMETERS

Prior to entering uniformity validation, it is necessary to have robust control of the preparation operations for the bulk filtration step. If the bulk filter is autoclaved or sterilized in place, this may result in retention of steam condensate on the filter, filter housing, and/or system piping, which in turn could result in dilution of the DS and increased variability in uniformity results. Additionally, the filter may be integrity tested prior to use (either prior to or after sterilization) or preflushed with buffer to remove potential extractables/leachables and equilibrate the filter. The buffer flush is especially important if the formulation buffer contains polysorbate or other agents which are known to bind to the filter. The buffer flush could alternatively be performed post autoclave/SIP of the filter, however, this adds complexity to the operation to ensure that the aseptic state of the equipment is not compromised during the flush. The integrity test procedure requires wetting of the filter with water. Although the majority of water is removed during the integrity test (diffusion or bubble point), there may be sufficient residual water retained in the filter. A good practice is to implement an in-process control point to ensure that the residual liquid from the integrity test flush, buffer flush, or steam condensate is reduced to an acceptable level prior to processing. This approach could be carried out in the form of an air-purge step, a filter-drying step (in an oven),or a simple comparison of the filter weight before and after preparation steps. The allowable residual fluid in the filter can be calculated based on the uniformity requirements. Such control measures provide confidence that the potential sources of dilution are removed or minimized prior to validation.

These controls are especially important to ensure that the DS meets specification limits in the event of reprocessing across the bulk filtration step (e.g., if the post-use integrity test fails). Table I highlights typical steps in preparation for the drug substance filtration operation. It should be noted that not all the steps shown in Table I may be required; some steps may be eliminated depending on the need for operational simplicity, for example, by minimizing manipulations such as the air purge or buffer flush to the filter assembly post autoclave. The final decision on the number and order of steps should ensure that the potential failure modes are adequately addressed while maintaining a robust and consistent process to meet the uniformity requirements.

In addition to the controls described above, it may be necessary to send a pre-determined product flush to drain in order to reduce dilution effects. While it is desirable to minimize this loss of product, it is imperative to ensure that yield optimization does not override the quality consideration for demonstrating the uniformity of the batch. Regarding uniformity performance parameters and acceptance criteria discussed previously, it should be noted that it is not necessary to demonstrate that the first drop emerging from the DS filter is absolutely identical/uniform with the rest of the batch as long as the lot release samples are not taken at this point. The quality consideration should take into account acceptance criteria that are scientifically justifiable and based on how the drug substance will be forward processed.

Another important consideration with respect to the product flush is the size of the filter and filter housing. While it is appropriate to size the filter with a safety margin so that it can process batches in a robust manner and in a reasonable time frame, oversizing the filter could result in challenges from a bulk uniformity perspective. One way to address this issue is to perform a prefiltration step with a larger filter to remove particulates which may foul the 0.2 µm filter. The prefiltration can be performed as part of the preceding unit operation so as to collect a clarified feed stream for the bulk filtration step. A further variation is to perform a batch-wise bulk filtration into a single collection vessel, thereby allowing for a buffer chase to maximize product recovery and also any dilution that may be necessary to achieve the target product concentration. This option could also be adapted into a continuous mode by the use of a surge tank between the filter and collection containers to avoid dilution effects. Following adequate mixing, the product may be dispensed aseptically into appropriate collection vessels. This obviates the need for the product flush, and thus maximizes product recovery during processing.

Other considerations to achieve uniformity at the bulk filtration step are the design and operation of the upstream feed vessel. Mixing studies should be used to establish set point and ranges for agitation speed and time to ensure thorough mixing of the contents to be filtered (e.g., top, middle and bottom of vessel). The design of the vessel is also important to ensure that dead zones and holdup volumes (both line and sample port) are minimized. This is especially important if dip tubes are present, which may be used for product introduction into the vessel and/or subsequent withdrawal for the bulk filtration step. Both examples may impact uniformity if lines are not efficiently flushed, or if hydrostatic pressure within the tube results in concentration changes over the course of the filtration. It is preferable to use separate routes for product introduction and to locate dip tubes such that they do not result in a dead zone within the tank. The vessel location and piping required to transfer the product from the feed vessel through the filter should also be evaluated to minimize holdup volume and ensure proper drainage (to account for condensate drainage following product transfer line steaming) in order to minimize the possibility of dilution.

BEST PRACTICES

Characterization studies to confirm that preparation procedures are adequate and robust should be performed prior to performing uniformity studies with product. These can be performed using buffers or salt solutions with pH and/or conductivity as convenient indicators to assess uniformity through filtration. Careful assessment of the buffer used for such studies is necessary to ensure that the buffer selected is a representative model to use, and parameters such as density and viscosity which could impact the kinetics of filtration should be considered. Figure 2 shows the result from one such study where a sodium chloride solution was used to determine whether a product flush would be required. As expected, the initial samples during the filtration step have a slightly lower conductivity but this quickly stabilizes to greater than 97% of the initial concentration. The results from replicate studies are consistent, showing that the operation is reproducible. An alternative strategy would be to utilize a representative protein surrogate, if possible.

It would be prudent to also perform a (non-GMP) engineering run with product prior to the process validation studies. This provides added assurance that the uniformity validation study acceptance criteria will be met. Figure 3 also compares the product profile with the surrogate salt filtration runs, confirming the results and conclusions drawn from the wet testing. If there is a potential for reprocessing at the bulk filtration step (e.g., as a result of failed filter integrity test post use or operational errors which may have compromised the aseptic nature of the batch), it is recommended to test the uniformity of the batch at the reprocessing step during the engineering run.

The standard practice in industry is to perform uniformity validation during the conformance runs (also referred to as process performance qualification, and historically referred to as process validation). Future process changes also require an assessment of the validated state of the step and whether revalidation is necessary to confirm that uniformity is not affected. However, since ongoing uniformity testing is not typically performed, subtle shifts or trends in the process would not be detected. The use of the filter weight checks as inprocess controls along with robust maintenance of operational parameters provide assurance that DS uniformity is maintained. It may be desirable to perform uniformity testing on a periodic basis to provide further confirmation. The approach of using the "percentage of feed stream concentration," described above, provides a simple means to confirm uniformity on an ongoing basis.

CONCLUSION

Drug-substance uniformity is an important consideration for the final step in the manufacture of API. Uniformity validation studies are necessary to ensure that the entire contents of the batch are homogenous and that the drug substance specification and stability samples are representative of the batch in terms of critical quality attributes. Using risk-based approaches and comprehensive process characterization studies, appropriate test parameters (e.g., protein concentration) and sample points (beginning, middle, and end) can be selected. Scientifically sound strategies which may be used in combination for developing uniformity acceptance criteria include those based on specification limits, measurement error, tolerance intervals of historical data and equivalency of sample means. Additionally, operational considerations, such as filter weight checks, pre-processing air drying, product flush, and the use of wet testing and/or engineering runs provide greater assurance of robust and consistent drug substance filtration and uniformity.

Sushil Abraham* is director of process development, Eric Rydholm, is principal engineer, and Phil Wagner is senior engineer, all at Amgen, Longmont CO, sushila@amgen.com.

PEER REVIEWED Article submitted: Oct. 20, 2010. Article accepted: Feb. 11, 2011.

REFERENCES

1. 21 CFR 211 (Government Printing Office, Washington DC), section 110.

2. G.B. Limenati, Analy. Chem. 6, 1A–6A, (2005).

3. S. Richter and A. Richter, Qual. Engin. 14 (3), 375–380 (2002).