Tuesday, December 23, 2014

Cleaning Validation Process

The primary aim of this article is to stress the importance of equipment cleaning as an official process. A typical continuum of an Equipment Cleaning Process (ECP) consists of:
  • Development of Cleaning Procedures by Research and Development groups (if it exists)
  • Validation of Cleaning Procedures by Technical or Quality groups
  • Application of Cleaning Processes by Operations/ Manufacturing organizations
Although all of the Cleaning Process and Validation written procedures stipulate that changes are covered by a change in management these procedures are rarely changed. A brief description above constitutes usual continuum for hundreds of pharmaceutical companies. However changes are seldom pursued as most of the companies perceive them as infringement upon validated process “magic.” What is meant here by “magic” is a documented study, assembled in the thick binder and put on the shelf never to be disturbed again that serves as a gold standard for future processes. However, most, if not all, validation activities are performed only a limited number of times while the processes they validate are run continuously. Therefore, it is impossible for a single validation exercise to gauge process variability in its entirety simply because of the limiting nature of a study.
A perceptive reader of this article may have noticed that those responsible for pivotal parts of the ECP continuum belong to different groups. Therefore an inevitable question, “Who is the Process Owner?” is typically and intuitively answered, “Operations or Manufacturing,” since they perform a majority of the continuum’s work. One might think, “They must be the owners since they clean equipment all the time.” Although, upon further examination it is clear that typically Operations or Manufacturing departments do not develop the process and do not qualify that it consistently delivers a quality result. Therefore, when cleaning fails it is typically assumed that it was not carried out correctly. Human error is often cited as a cause of deviations and nonconformances. Most of the time, despite extensive global regulations and a voluminous technical literature library on the subject, a failure of a Cleaning Process is not a human error but a failure to recognize the Cleaning as an official process and treat it as such. Instead of pursuit of process understanding, these failures cause organizations to perceive ECP as a “necessary evil"—something to tolerate mostly due to regulations. Many personnel may try to avoid it altogether and might argue that it is not a value added activity. However, it is obvious that this kind of philosophy is wrong and Cleaning must be recognized as an important and necessary part of pharmaceutical manufacturing. If one does not develop an appropriate method for Cleaning Process residuals, does not appropriately validate this method, and does not assure that it is still viable throughout the lifecycle of the product, one is adding another unknown variable into an actual manufacturing process. As stated by Deming, “uncontrolled variation is the enemy of quality.”
Therefore, the goal of this article is to help practitioners in development, utilization, and maintenance of Cleaning Validation programs so that they can reduce process variability thus answering the question—How Clean is Clean in Drug Manufacturing? To achieve this goal we will touch upon an important aspect of Cleaning Validation that gained traction in the last several years due to the implementation of risk-based lifecycle approach to Cleaning Validation built on principles of ICH Q8, 9, 10 Guidance, FDA Guidance for Industry: Process Validation, as well as EU Annex 15: Qualification and Validation (recent draft).
The subject we will discuss is development of limits for the process residue. Knowledge of the limits-setting strategies should help in gauging one’s Cleaning Validation program.

Establishing Limits

The subject of soil residue limits setting is of an utmost importance because it is a measure of the Cleaning Process effectiveness and consistency. We measure the success of a Cleaning Validation study by assuring that we meet predetermined criteria based on removal of the soil to a level below an established limit. Therefore establishing limits is one of the pivotal steps in the Cleaning Validation continuum. Although there are many sources on this topic and we can specifically mention a few excellent references for various methods of setting soil residual limits as well as history of the subject, our goal would be to employ science and knowledge into this exercise. First, we will briefly talk about Health-Based Exposure Limits since this subject has been debated for a few years and readers may benefit from some level of demystification. We will only summarize a few important points to consider when evaluating Health-Based Exposure Limits. There are several major terms being currently used globally for these limits. They are listed in Table 1 along with their respective sources and some notes that describe their intentions and use.
Table 1. Health-Based Exposure Limits Terms
Zoom In
It is important to note that ADE and PDE are often referenced by regulators. However, if ADE has been routinely used in the United States, PDE is been consistently referenced by the European Guidance. These two terms, although similar, have considerable differences. ADE is extrapolated from NO[A]EL while PDE is typically based on extrapolation from NOEL. The difference between NO[A]EL and NOEL is very important. Table 2 compares descriptions for these two levels.
Table 2. NOEL vs. NO[A]EL
Another reference to consider with regards to the difference between two levels is FDA’s view, which was cited with respect to comments raised by industry for the “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” Guidance for Industry. In their response, FDA said “the NO[A]EL is not the same as the no observed effect level (NOEL), which refers to any effect, not just an adverse one, although in some cases the two might be identical.”6 FDA went on to state, “the definition of the NO[A]EL, in contrast to that of the NOEL, reflects the view that some effects observed in the animal may be acceptable pharmacodynamics actions of the therapeutic and may not raise a safety concern.”7
Cleaning Validation practitioners should consider these differences and utilize a toxicologist or a person with adequate training in pharmacology and toxicology to develop and document Health-Based Exposure Limits Assessments for soil residual limit calculations.
Although undisputedly important, the Health-Based Exposure Limits should not be the only ones utilized for measuring of a Cleaning Process. In order to gain a thorough understanding of the level of cleanliness needed, it is recommended to utilize the limits-setting practice using multiple levels. These levels are summarized in Table 3.
Table 3. Risk Levels for Setting Cleaning Validation Limits
Zoom In
Usually, for pharmaceutical drug products, Health-Based Exposure Limits would be the largest from these three levels unless there are toxicological hazards associated with an Active Pharmaceutical Ingredient (API). The Visible Limits are typically very similar to historically used 1/1000th of the lowest dose of the residual soil in the maximum daily dose of the next product or 10 ppm of the previous API in the next product manufactured. An illustration of this relationship is shown in Figure 1.
Zoom In
Figure 1. Typical Visible Residual Limits, MACO Limits (Cleaning Validation Achievable), and Health-Based Exposure Limits
It is important to note that MACO 1 ppm limits are readily achievable by most validated Cleaning Processes. In addition, these Cleaning Processes are typically found to be statistically capable. Therefore it is highly helpful to use statistical process capability analyses for evaluation of these results.9


A thorough knowledge and utilization of riskbased limits setting techniques shall help Cleaning Validation practitioners determine with a high certainty the level of cleanliness they can achieve in day-to-day production, thus answering question, “How Clean is Clean in Drug Manufacturing?” The bottom line is that set ECP residual limits “should be logical based on the manufacturer’s knowledge of the materials involved and be practical, achievable, and verifiable.”10 They should be set with an input and buy-in from all groups engaged in ECP continuum. Upon setting the limits an organization should establish the Process Owner (typically a team responsible for validation activities) to ensure that the process is capable to meet these limits throughout its lifecycle. In addition, it may be helpful to review the process performance status such as adherence to specifications (set limits) with all of the continuum groups on a periodic basis. These sessions should help communication between the groups while nourishing knowledge management culture elevating ECP to a new level of understanding and respect.

Variability and Test Error with the LAL Assay


The Limulus amebocyte lysate (LAL) assay is the compendial test for the examination of bacterial endotoxin in pharmaceutical products (as described in USP chapter <85>), in-process material, and pharmaceutical grade water.
With any biological tests, measurements are susceptible to variations in analytical conditions. Here the LAL assay has a relatively high level of variability even for a biological assay.1 This variation derives from 3 principle sources: reagents, product, and method.2 This paper examines some of the reasons for LAL test variation, focusing on photometric methods (chromogenic and turbidimetric), and considers how variation can be assessed through good laboratory quality control.

LAL Test Variability

Different aspects of the LAL test can cause variability. These include the LAL reagent, endotoxin control standard, standard curves, and dilution error. These are examined in turn.

LAL Reagent

The LAL reagent is a contributor to assay variation for the following reasons:
  • The LAL reagent (lysate of the horseshoe crab Limulus polyphemus) is of biological origin. It is a complex mixture of enzymes and co-factors. The extract is relatively crude mixture and is not a single purified enzyme. This means that the enzyme activity cannot be determined exactly for each lot of lysate manufactured.
  • The manufacturing process includes the addition of buffers and detergents which contribute a further source of variability.
  • The enzymatic activity of each lot of LAL is assessed by the manufacturer using Reference Standard Endotoxin (RSE, supplied by the USP). The LAL sensitivity is assessed by performing a 2-fold dilution series. The RSE used to characterize the lysate is not readily available to all test laboratories because of its rarity and cost. Laboratories normally use Control Standard Endotoxin (CSE). The potency of CSE is determined by the lysate supplier assessing the CSE against RSE. This adds a potential area for test variation.

Bacterial Endotoxin

The endotoxin used in the assay can cause variation. This is because:
  • The endotoxin used to prepare the CSE used in laboratories is from a purified strain of Escherichia coli. CSE is presented as a highly purified lipopolysaccharide free of most detectable contaminants (such as proteins). The CSE contains additional stabilizing fillers like starch, human serum albumin, and polyethylene glycol. However, environmental endotoxin is not purified and normally takes the form of a macromolecular complex of lipopolysaccharide, cellular membrane proteins, and phospholipids which are shed by Gram-negative bacteria during growth and death. Thus there is variation in assaying environmental endotoxin against purified endotoxin standards.3
In addition, although the LAL test is specific for endotoxin, it will detect only the Lipid A portion of the endotoxin molecule which is available to activate the lysate (the activation of the clotting cascade, the Factor C pathway, is described below).4 The Lipid-A portion of the endotoxin molecule may form aggregates which are not fully dispersed and therefore not homogenous enough to allow for accurate total measurement.
Thus, a sample which detects endotoxin may not show all of the endotoxin in the sample, for this depends upon the amount of Lipid-A available. Therefore, samples which detect endotoxin may be underestimates. Furthermore, a sample which detects endotoxin may not demonstrate the same level of endotoxin when repeated because the availability of Lipid-A may alter as the chemical nature and stability of the sample changes over time.
  • It should also be noted that the toxicity and reactivity of different types of environmental endotoxin differs depending on the biological activity of the Lipid-A molecule for different bacterial species.

LAL Test Variability

The LAL assay has an inherent variability of 50% to 200% (or one 2-fold error either side of each endotoxin standard). Variation arises, for kinetic assays, from the slope of the endotoxin standard curve.5
Test variation can arise from a range of test inputs, including:
  • Test tubes
  • Disposable pipette tips
  • Micropipette tips
  • For the above, plastics used in the performance of the BET (eg, microtiter plates, plastic dilution tubes) are often not made specifically for endotoxin testing
  • Aseptic technique
  • Assay technique
  • Variations in pipetting
  • Variations in preparing control standards
  • Variations in preparing dilutions (which is magnified if the error occurs with the first dilution in the series)
  • Dilutions stored over the longer term will show change. Variable factors include temperature, vessel composition, dilution range, and volume of the dilution)
  • Cross contamination
  • Product or sample interference
  • Sampling containers
  • Sample storage times and temperatures
  • LAL instrument/module variability–different instruments may give different results;
  • Presence of endotoxin in product (where endotoxin molecules behave differently or where the availability of Lipid-A varies)
  • Addition of buffers to stabilize pH
  • Ancillary solutions may not be free of endotoxin.
  • Some of the above relate directly to the practices of the testing technician (such as preparing dilutions, pipetting, weighing raw materials, and aseptic technique)

Endotoxin Concentrations

The significance of error also increases as the endotoxin concentrations used for a standard series become smaller. For example, with a standard curve of 1.0 to 0.1 EU / mL errors of 50% to 200% will have a lesser impact than a standard series of 5.0 to 0.005 EU/mL, based on the smaller value of the last endotoxin concentration in the standard series. It is perhaps for these reasons the acceptable spike recovery of test controls listed in the pharmacopeia is 50% to 200%.

Dilution Errors

Errors can occur with test dilutions, especially with those relating to the dilution of endotoxin and creating a standard curve. To avoid the possibility of dilution error arising from the construction of the standard curve, it is recommended that the following controls are put in place:
  • The dilution series should be qualified before each lot of endotoxin or lysate is released for routine use, by 3 technicians who verify the dilution series 3 times each. A similar exercise is undertaken for each new technician who is trained in the test.
  • For routine assays, the dilution series should not vary between tests (that is, the same types of dilutions are always undertaken).
  • The starting concentration of endotoxin should always begin with the same value (this is normally with 1000 EU/mL). Endotoxin standard curves are constructed from the same starting concentration of endotoxin: 1000 EU/mL. This is verified by reviewing the manufacture’s Certificate of Analysis and from undertaking confirmatory tests of the manufacture’s certificate by comparing the in-use Control Standard Endotoxin against Reference Standard Endotoxin (both a purified extract of E. coli O113:H10).
Nonetheless, with the above in place, a dilution error can still occur where a mistake is made on the part of the technician conducting the test.

Standard Curve Linearity

Standard curve consistency is an important feature of the LAL test. A change of only 1% in y-intercept for a linear standard curve can result in a 30% to 35% change in endotoxin determination. Hence a sample with a known 10 EU/mL can read 13.5 EU/mL, not because of a change in the endotoxin content of the sample, but because of a shift in the y-intercept. An important means to control variability in the turbidimetric LAL test is to keep an eye on the onset (reaction) times. Seemingly small changes in these onset times result in changes to linearity, slope, and y-intercept that can have a significant effect on the test result.6

Assessing Variation

Whilst steps can be taken to reduce variation, the principles of good quality control dictate that a laboratory should have an oversight and a means of assessing if tests are satisfactory and whether variation has been sufficiently excessive as to cause concern. One means of doing so is through reviewing the coefficient of variation (CV).

Applying the CV

CV is a measure of precision. The precision of an analytical procedure is the degree of agreement among individual test results (or, in assay terminology, the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single, homogenous volume of the biological matrix).7 The CV is the standard deviation expressed as a percentage of the mean. As the mean between different samples increases, then the CV can be used to measure the variability.
Commonly, CV is expressed as a percentage between test replicates (%CV). This can be applied to standard curve points and to test sample replicates. With LAL, the common requirement is for the CV to be ≤10% or ≤25% (depending on the requirement set by the lysate vendor). The lower the %CV, the closer the level of precision between the different test replicates is (the ‘scatter’ from the mean is relatively small).
There are different approaches to the calculation of CVs. These relate to different ways of calculating the standard deviation. Once the approach for calculating the standard deviation has been adopted, the CV becomes expressed as the ratio between the standard deviation and the mean.
From this, the %CV can be calculated thus:
100% x (standard deviation/arithmetic mean)
From the above it can be seen that the CV is converted into a percentage by multiplying the obtained number by 100 to produce the %CV.8
In assessing LAL tests, %CV values calculated on measurements of EU/mL typically increase for lower concentrations of endotoxin, in that values of 10% can often be obtained for higher concentrations of endotoxin, whereas values of between 10% and 30% are obtained for lower values of endotoxin (typically towards the end of the standard curve and close to the limit of detection).
It should be noted at this point that different LAL suppliers have different acceptance criteria for the LAL test and calculate their %CVs, through software packages, in different ways. Some, for example, calculate the CV based on the results of obtained in Endotoxin Units per millilitre (EU/ mL); whereas others calculate coefficient of variations based on the sample onset times (in milli-absorbance units).9,10

Detecting Dilution Errors

In addition to the coefficient of variation, dilution errors also require assessment. In order to check that the LAL assay has been performed correctly, the reaction time for the highest endotoxin concentration point on the standard curve should be examined to ensure that it lies within an expected time range in seconds. This is important because seemingly small changes in these onset times result in changes to linearity, slope, and y-intercept that can have a significant effect on the test result.11
For this, the onset times for the starting endotoxin concentration are examined (such as 5.0 EU/ml). This requires a study of historical data in order to establish the typical range. For greater accuracy, it is recommended that the first 100 tests performed using the endotoxin are assessed. If an error occurs with the preparation of the dilutions, then the onset time would fall outside of the expected range.
Examining the onset time is an important indicator of assay error as it directly relates to the way in which the photometric LAL test method works. With the kinetic-turbidimetric or chromogenic LAL test method, lysate (when aliquoted into reaction tubes) reacts with any endotoxin present in an aliquoted sample or within a standard curve dilution.12 The reaction which takes place is one of turbidity or color change as measured against time. The faster the time taken to reach a turbidity threshold (measured in milli-absorbance units at a pre-set optical range), the greater the endotoxin concentration. This onset time range not only varies depending upon the level of endotoxin, it will vary for different lots of lysate and Control Standard Endotoxin.
It is important that the onset time falls within the correct range because it establishes that the correct dilutions have been performed. It is possible, for example, that if a test was diluted incorrectly, the correlation coefficient and the reported endotoxin values would still appear to be correct. This is because the correlation coefficient is the line of best fit between actual and expected values and the software that interprets this data and produces an estimated endotoxin concentration by extrapolating from the standard series.13
Figure 1.
One way by which the range for the time taken for the optical density of the starting concentration to reach the required threshold can be calculated from the second standard deviation from the mean. Given the inherent inaccuracy of the LAL test (which is commonly accepted as ±25%), and to incorporate a level of standardization with approaches taken to other biological assays, the second standard deviation is arguably the appropriate measure. The standard deviation is a measure of how much individual elements tend to deviate from the average (mean). It is calculated as the square root of the variance (as shown in Figure 1).
The use of 2 standard deviations from the mean indicates that 95% of the values fall within the upper and lower ranges. The 5% outside of this are taken to represent atypical values.
After the completion of each LAL test, the onset time recording the highest endotoxin concentration (the start of the curve) are compared against this range and the test is only deemed to be acceptable should the measured onset time fall within this range. If a dilution error had occurred, then the onset time would have fallen outside of the expected time range.
by adopting 2 standard deviations from the mean, any test values which fall outside of the calculated range are said to be atypical values and not representative of the normal population. On this basis, the LAL test requires investigation. As a way of a further check on technician performance, the onset times for each technician’s standard curve can be trended and examined by the area supervisor.

Standard Curve Correlation Coefficient

An additional check can be made on the linearity of the standard series. With this, the correlation coefficient of the standard curve should be examined for each test (where the requirement is for a correlation coefficient of 0.980 or greater). This check is required by the pharmacopoeia for each test run.
Standard curve consistency is an important feature of the LAL test. A change of only 1% in y-intercept for a linear standard curve can result in a 30% to 35% change in endotoxin determination. So, a sample with a known 10 EU/mL can read 13.5 EU/mL—not because of a change in the endotoxin content of the sample, but because of a shift in the y-intercept.6

Negative Controls

Negative controls should be run for each test. The negative controls are samples of the water used to construct the standard curve. The negative control samples will indicate if any contamination has occurred during the preparation of the endotoxin series. The controls are required to have an endotoxin level below that of the lowest endotoxin concentration within the standard curve (for the routine standard series, this is 0.005 EU/mL). This check is required by the pharmacopoeia.


This paper has considered some of the sources of variation which affect the LAL test. These are important for the laboratory user to understand, especially for the design of the assay and with the investigation of test anomalies. The article has also considered one key measure of variability: the coefficient of variation. This is an important check for the laboratory supervisor to include when reviewing test results.


  1. Williams KL. Endotoxins, Pyrogens, LAL Testing and Depyrogenation, 2nd edition, CRC Press: Boca Raton. 2007.
  2. McCullough KC and Weider-Loeven, C.: ‘Variability in the LAL Test: Comparison of Three Kinetic Methods for the Testing of Pharmaceutical Products’, Journal of Parenteral Science and Technology. 1992;44:69-72.
  3. Brandberg K, et al. : Conformation of lipid A. the endotoxic center of bacterial lipopolysaccharide. Journal of Endotoxin Research. 1996;3:173-178.
  4. Moser K. Playing Hide and Seek with Endotoxin, LAL User Group Newsletter. 2009;3(2),1-5.
  5. Kumar H. ‘Variability in the Bacterial Endotoxin Test or LAL Test’, Endosafe Times, Charles River Laboratories, USA. 2007.
  6. McCullough K. (2008): Laboratory Variability, LAL Users’ Group Newsletter. 2008;2(3): 6-7.
  7. Brosnahan K. “Understanding Correlation Coefficients and Coefficients of Variation in Photometric LAL Testing”, LAL Update. 2006;23(2):3-5.
  8. Richardson K. and Novitsky, T.J. ‘Simple Statistics for the LAL User – Standard Deviation, Repeatability, Reproducibility and a Clarification of the Coefficient of Variation’, LAL Update. 2002:20(4).
  9. Lindsay GK, Roslansky PF, Novitsky TJ (1989).S ingle-step, chromogenic Limulus amebocyte lysate assay for endotoxin, J Clin Microbiol. 1989;27(5): 947–951.
  10. Górny RL, Douwes J, Versloot P, Heederik D, Dutkiewicz J. Application of the classic Limulus Test and the Quantitatibe Kinetic Chromogenic Method for Evaluation of Endotoxin Concentration in indoor air. Ann Agric Environ Med. 1999; 6:45-51.
  11. Tsuchiya M. “Biases in the Bacterial Endotoxin Test”, BioProcess International Industry Year Book 2010-2011:144-145.
  12. Guy D. “Endotoxins and Depyrogenation” in Hodges N. and Hanlon G. Industrial Pharmaceutical Microbiology: Standards and Controls. Euromed, 2003:12.1–12.15.
  13. McCullough K. ‘Back to Basics: Where Did My Standard Curve Come From?’, LAL User Group Newsletter. 2009:3(2):6-8.

Forensics in QbD: Addressing Foreign Particulate Matter Investigations

Foreign particulate matter (FPM) can affect product efficacy, but more critically, it can affect product safety leading to recalled product or regulatory action (eg, FDA warning letter). FPM investigations can be costly and sometimes inconclusive which complicates decisions about product release. There are ways to streamline these investigations through an approach in line with Quality by Design (QbD). Particulate programs couple the right characterization technique, a proven method for historical data organization, more enhanced manufacturing process understanding, and risk-based decision making. These attributes of a particulate program come together to enhance the quality of FPM investigations in a manner that is cost effective, efficient, and, most importantly, conclusive.

Program Requirements

The ideal particulate program is comprehensive and able to grow with and into the needs of an organization. Being flexible and versatile allows the program to handle diverse FPM investigations and provides a knowledge bank to query when repeat issues arise. The program should also be cost effective and sustainable, benefitting all levels of staff within the organization. Above all, however, the FPM program should be conclusive, leading to closure for each FPM investigation.

Program Components

Figure 1. Common Particulate Program Components
Program components may differ across organizations in order to meet requirements. Figure 1 lists those commonly seen.

Initial Components

Regulatory requirements necessitate product inspection, and FPM is identified during the initial portions of the program. The next step is removing FPM from the process. Sampling can be as simple as separating finished product containers or as complex as retrieving a residue from the bottom of a 20,000-L tank. Next, the FPM must be isolated from the matrix material. There are numerous techniques for isolation such as pipetting, filtering, or extracting. The most appropriate technique will depend on the specific set of circumstances. Once isolated, FPM should be rinsed with particle-free water to ensure it is free from the matrix material.
Isolation requires the most practice and specialized tools because FPM can be as small as 20 μm, which is beyond the visualization limits of the human eye. Therefore, a stereomicroscope must be used to facilitate isolation. Particle sizes range typically from 50-300 μm, but can be as large as a few millimeters (easily seen by the human eye). Using tools, such as a tungsten needle, to manipulate small quantities of material under the stereomicroscope takes practice. Isolation poses a risk to sample integrity through factors like static from the outside environment, by the type of tool that is being used, or by loss if too much pressure is improperly applied with tools. Residual product can also affect the way FPM behaves once it has been exposed to the environment. Less experience with isolation increases risk. It is possible to isolate and evaluate particles smaller than 20 μm, but preparation and characterization strategies are different from those typically used.


Categorization allows for flexibility and versatility. During this portion of the program, categories or bins are identified for later use. We categorize many different particle attributes including: morphology (flake, fiber, residue, speck, globular, etc), luster, transparency, color, quantity, mobility, and buoyancy. Additional attributes could be important to particulate found in a manufacturing process, but the relevance of any attribute category will depend on specific processes and circumstances. It is here that optical inspection (or assisted visual inspection) becomes important.
Often, specific particle attributes of FPM can only be visualized with the aid of the stereomicroscope. It is possible to have categories from both visual and optical inspection used independently or in concert. Establishing these categories enables trending that assigns value to the FPM data. For example, trending morphology may be of interest. How many fibers have been encountered in production process A? What is the trend in terms of color and a time constraint? How many blue particles were found last year? Is there a particular time of year when one particle type is more prevalent? Categorization is also key in utilizing tiered approaches for cost and efficiency, which are discussed later.


Current methods of characterization, such as visual inspection, microscopy, and light obscuration, can support the program up to this point. The main issue is that the information these methods provide, in and of itself, is not adequate enough to trace back to root cause. Figure 2 illustrates an example. All of the particles would appear similar to current methods. The particles are relatively the same size and shape and they have very similar color and luster. However, there are four different sources. Particles A-D are polypropylene, polystyrene, polyethylene terephthalate, and silicone, respectively. Therefore, there are four different root causes to consider. Particulate identification becomes key for root cause determination.
Figure 2. Particles exhibiting similar attributes but of different source materials
Figure 3. Particulate Characterization Regimen Analytical Techniques
Now that a characterization need is identified, requirements are reviewed. First, this regimen must be economical in its need for sample material. Quite often particles are less than 100 μm, so traditional bulk analyses cannot be employed. Second, the regimen must be efficient to meet the goal of quick investigation resolution. Last, the regimen must be capable of identifying numerous types of material. Many different types of organic and inorganic materials are utilized in a manufacturing process. An established regimen already exists that can satisfy these requirements. This regimen is forensic trace evidence characterization. This regimen is accustomed to small sample size as it is utilized for single fiber transfers, paint smears, and other types of transfers involving miniscule amounts of material. The regimen is well established and efficient, having been honed in the forensic community for years. Finally, the regimen can identify a very diverse set of materials in both the organic and inorganic realm. The techniques that are coupled together in this regimen and the data they provide are shown in Figure 3.
This characterization component allows the program to be comprehensive (clarifies the big picture) and conclusive (leads to root cause) which brings flexibility. A large set of complex data now becomes a living entity that is easily reduced to glean necessary information. Sustainability is also positively impacted because once established, the program can be maintained by individuals with varying technical backgrounds. The tradeoff for this conclusiveness is cost. Quite often, characterization is the most costly component of the program.

Case Studies

These case studies demonstrate the use of FPM program components and include real-world data and scenarios. They lead into a discussion about how tiered approaches can save time and cost in investigations.

Case Study 1 – Stability Study Contaminant

FPM was observed during a stability study. It was sampled by sacrificing one of the stability study containers and further isolated by vacuum filtration due to the small size and fragile nature of the particulate. Particulate were categorized as needle-like and were found in high quantity. They were also noted to be clear and crystalline in nature. Characterization identified this material as a polymer additive. After further discussion with the container vendor, it was revealed that the polymer additive matched one that was used to manufacture the containers. It was theorized the high heat of the stability study caused the additive to leach into the product. The issue was resolved by switching to a new container for subsequent studies that could withstand the required temperature. This investigation was lengthy and costly due to the fact that no particulate program was in place. A great deal was understood about the product, but not what could contribute to FPM in the product. Consequently, many potential sources were investigated until the polymer additive was identified by the vendor.

Case Study 2 – Finished Product Contaminant

A finished vial of parenteral product contained FPM and was reserved for sampling. The single particle was isolated by direct removal with a stainless steel scalpel blade. Categorization yielded a color and texture like that of the septum material accompanying the vial (gray and malleable). The container closure components of this manufacturing process were well characterized, but there were few other process components included resulting in only a partial program. From characterization, the particle was identified as butyl rubber with silicate fillers which was consistent with the septum material. A review of the process was initiated to identify other potential sources of butyl rubber. After this review, it was concluded that the vial septum was the only potential source of the FPM. An additional forensic technique, fracture matching, was employed to match a gouge in the septum with the shape of the particulate and added to the certainty that this particulate originated from the accompanying septum. A resolution was identified when a study of administration technique showed that personnel puncturing the septum were doing so improperly. Additional training was provided to alleviate the problem. By having a partial program in place, the manufacturer bypassed the need to characterize container closure components for this particular investigation.

Case Study 3 – Purification Contaminant

FPM was easily sampled directly from the manufacturing process with simple tools. No further steps were required for isolation. The numerous particles were categorized as white and fibrous. A partial program was also being utilized for this manufacturing process where some process components were fully characterized and some were not. This necessitated the characterization of the FPM which was identified as polypropylene. However, none of the components in the partial program were composed of polypropylene. An investigation was initiated to identify sources of fibrous material outside of those characterized in the program. Eventually, it was revealed the fibrous material was linked to a cleaning component. This component had been substituted and was not fit for the intended purpose. The partial program had been successful in previous investigations in identifying root cause, but failed to facilitate this particular investigation. It did however, cut back on the number of source materials that required investigation.

Case Study 4 – Intermediate Production Contaminant

A filter membrane within an intermediate production step contained FPM. The entire filter was sampled and the particulate isolated from the membrane itself. Several particles were categorized as very hard, brittle, and black with a luster. A comprehensive particulate program was established for this manufacturing process and there were several possibilities that fit the categorization attributes (ie, several sources for very hard, brittle, and black particles). The material was characterized as silicon carbide and from this, it was immediately known to source from a gasket seal. Essentially, no additional action was needed and the investigation concluded. This seal was known to wear and the amount of particulate observed was consistent with the wear typically seen at this point of the seal’s lifecycle.
From these case studies and in general, the amount of time it takes to conclude an investigation is proportional to the breadth of the particulate program in place (refer to Figure 4 for relative time and costs). That is, Case Study 1 took the most time. Case Studies 2 and 3 were shorter, but could be made more efficient, and Case Study 4 was most efficient due the comprehensive program that was in place. There are some upfront costs associated with program initiation; however, with tiered approaches, comes a possibility to balance these costs against the return of much faster investigation times.
Figure 4. Case Study Summary

Tiered Approaches

Particulate program requirements include: comprehensiveness, versatility, flexibility, and conclusiveness. These requirements have been satisfied, but the cost effectiveness requirement remains. The flexibility of the program is an advantage for building tiered approaches which can be used to address cost effectiveness.
Between the program components of categorization and characterization, there is an opportunity to make decisions about whether or not it is necessary to proceed with characterization. Once pertinent particle attributes have been identified and an established program has historical data to reference, this knowledge base can be combined with a thorough understanding of the manufacturing process. This comprehensive body of knowledge regarding the propensity for FPM can then reduce the need to escalate all particles into the characterization component. This body of knowledge will also help indicate at which manufacturing process steps the particulate profile of the product should be studied and allow boundaries to be set in terms of what to include in the program as a reference materials.
A risk-based approach is then relied upon at the decision point to determine whether the program needs to continue. Risk tolerance of the organization as well as situational risk (eg, healthy versus sick target population, child versus adult population, etc) dictates a forward path. When indications are within risk tolerance, characterization is not necessary. The most cost invasive part of the process is then reserved only for necessary instances. In order to achieve this cost effectiveness, however, the comprehensive program must be built.
Going back to Case Study 2, one could envision a full program showing that no butyl rubber sources aside from the septum existed in the process. It would also be known through establishment and routine use of the program that each time a gray, malleable particle is seen, there is confidence the material is butyl rubber. This collective information is used to eliminate the need to characterize the FPM. Each time a gray, malleable particle is encountered, it is within the risk tolerance to assume consistency with the septum material.
Case Study 4 provides an additional example. One could envision in this case the instance where no other particles matched the categorization attributes of the encountered FPM. The investigation would essentially be concluded at categorization. Time and costs are eliminated from the process. The most costly part of the program is used only when necessary and reserved for situations like anomaly particles or particles with similar categorization attributes.

FPM in the Lifecycle

The program and its components have been discussed thus far. How is this program proactive versus reactive and how does it fit into the product lifecycle? Traditionally, FPM investigations are linked to latter portions of the product lifecycle. Something is encountered in manufacturing and reacted to by initiating an investigation. To be more proactive, forethought should extend into earlier portions of the lifecycle even as far as clinical trials. This creates the mindset to consider particulate before it becomes a problem.
Figure 5. Example Product Lifecycle and End Product Specification
The guiding factor and starting point will be the end-product specification. For example, in parenterals, the guiding factor would be that the product is essentially free from visible particulate (Figure 5). To support this specification, evaluate the impact to the product from actions at every process step. Evaluate how the decisions made during scale up can impact FPM loading and what parts of the manufacturing process have the propensity to put particles into products. What process components are used? Do these have a history of generating particulate? Are there moving parts that can shed? How is the integrity of the component tested? Decisions about raw materials can also impact final product specification. How much FPM is contained in excipients from Vendor A versus Vendor B? Can this FPM contribute to FPM in the final product? A deeper understanding of impact upstream contributes to the understanding for propensity of FPM in final products.
This task can sound overwhelming and daunting; however, the natural segments of the process can be used to delineate how to begin addressing it. A certain level of process understanding will exist and is then built upon through the natural lifecycle. This is the approach to addressing FPM that is very much in line with QbD.


It has been established that the current methods for addressing FPM fall short in root cause determination. Without root cause, investigations become very cumbersome and expensive to manage and close. Clear benefits arise (flexibility, versatility, sustainability) when particulate investigations are conducted as part of a larger particulate program. Tiered approaches allow investigations to be completed efficiently. And the comprehensive nature of a full program helps to ensure particulate investigations arrive at closure. These benefits ultimately allow high quality products to reach patients faster and more cost effectively.


wes would like to thank Ms. Elizabeth Wolff for her assistance with this article.

Role of Environmental Monitoring and Microbiological Testing During Manufacture of Sterile Drugs and Biologics*


The microbiological quality of drugs and biologics is necessary for their efficacy and patient safety, because microbial contamination of drugs causes immediate adverse effects on patient health in terms of morbidity and mortality,1-3 as well as long-term adverse effects, such as cancer, autoimmune, and other diseases. Additionally, microbes can alter the chemistry and pharmacology of drugs, with a potential adverse impact on their effectiveness due to the breakdown of the active ingredients as well as on their safety due to the toxicity of potential degradant products. Therefore, control of microbes in drugs is essential, either by assuring absence of microbes in sterile drugs that are administered parenterally and applied to sensitive tissues or by controlling microbial bioburden to appropriate levels for nonsterile drugs that are administered to regions rich in microbial flora with physical or immunological barriers to infections. Table 1 lists major differences between sterile and non-sterile drugs. For sterile drugs, microbes are essentially eliminated by terminal sterilization (heat or irradiation of final containers) or by employing an aseptic manufacturing process where terminal sterilization is not possible, specifically for most biologics. Assurance of the absence of bacterial, yeast, and fungal contaminants is provided by the sterility test for sterile drugs.4 For non-sterile drugs, bioburden due to aerobic bacteria, yeast, and fungi and absence from objectionable microorganisms, as required, is controlled to appropriate levels based on product attributes, route of administration (oral, intranasal, topical, anal, vaginal, etc) and target patient population (neonates, infants, elderly, immunocompromised, healthy population, etc). Non-sterile drugs are tested for total aerobic bacteria, yeast, and fungi by the bioburden or microbial limit test5-7 and for the absence of objectionable organisms,6-11 as required (Table 1).
Table 1. Major Differences between Sterile and Non-Sterile Drugs and Biologics

Limitations of Microbiological Testing

Microbiological testing plays a significant role in assuring the appropriate quality of drugs. However, the paradigm of final product testing, particularly for microbiological quality, is shifting, because testing alone does not provide complete or absolute assurance for control or absence from microbes (eg, bacteria, fungi, mycoplasma, and viruses). Additionally, the reliability of microbiological testing depends upon the selection of appropriate methods that are “Suitable for Intended Purpose” and an adequate number of samples taken at appropriate stages of manufacture.12 For example, to provide an absolute assurance for the absence of microbes in a product, the whole product will be required to be tested for sterility. After the test, there will be no product for actual therapeutic use.

Building Microbiological Quality into Drugs

Microbiological quality needs to be built into the drugs by understanding the sources of contamination, environmental conditions, and product attributes that support growth of microbes. Microbiological quality for sterile drugs is assured by employing a robust environmental monitoring (EM) program, appropriate microbiological testing at various stages or intermediate products during manufacture, including the final drug product (DP) and using validated manufacturing processes (eg, aseptic manufacturing processes, container closure studies, media fill studies, etc). During routine manufacture of sterile drugs employing aseptic manufacturing processes, EM is an essential and critical component to demonstrate the state of control of the facility, providing information on the microbial quality of manufacturing and testing environments. This is an important element for sterility assurance of sterile drugs. There are a number of guidance documents and regulations on the EM aspects of manufacture of sterile drugs.13-15 Microbiological quality of nonsterile drugs is important, too, and can be assured through selection of appropriate controls through a risk analysis process. Many sterile drugs have certain components or intermediate products that are classified as non-sterile and are manufactured like non-sterile drugs. Therefore, understanding the risk of introduction of microbes and their products (such as toxins and proteases) during manufacture of non-sterile drugs, and intended use of the product in a target population (such as use of vaccines in healthy individuals) are important considerations in choosing a manufacturing process—sterile or non-sterile. There are expectations and a need to control and monitor the environment for manufacture of non-sterile drugs, intermediate products, or components.6,8,16,17 However, there is not much guidance or clarity on regulatory expectations on the EM program for non-sterile drugs. Recently the United States Pharmacopoeia (USP) drafted guidelines to monitor the environment for manufacture of such drugs.18 These guidelines describe a risk-based approach to control microbes for manufacture of non-sterile drugs.
In this article, the role of EM and microbiological testing in eliminating or controlling primarily bacterial, yeast, and fungal contaminants during manufacture of drugs and biologics—specifically vaccines—is discussed. Control and testing for adventitious viruses, mycoplasma, residual live viruses or bacteria, and other aspects of microbiological testing critical in the safety of biologics, are not covered in this article. Recently, there have been significant concerns and discussions about the sterility assurance of drugs formulated by compounding pharmacies and microbial control during such operations due to a number of adverse events, including deaths from use of fungal contaminated methylprednisolone injections.3 This article does not cover microbiological quality of drugs made by compounding pharmacies. The USP has several chapters on controlling microbiological quality of such drugs.19-22

Challenges in Assuring Microbiological Quality for Biologics

As discussed above, microbiological quality of drugs and biologics is critical for their safety and effectiveness. But biologics, particularly vaccines, pose unique and complex challenges in achieving microbiological quality (Table 2). Biologics, as per their definition, are made from starting materials that are biological in nature and support microbial growth during the manufacturing process, creating challenges in maintaining sterility or purity of the desired organism. Many biologics are made in embryonated eggs, animals, and cells of avian, mammalian, or insect origin, collectively referred to as the substrate, which may contain inherent adventitious agents and support the growth of microbial contaminants. Starting materials, such as seed viruses or bacterial seed stock cultures, may consists of pathogenic or attenuated bacteria or viruses posing a risk to the operators, environment, and the final product due to presence of residual live bacteria or viruses and active toxins. Further, several raw materials, such as growth media, fetal bovine serum, trypsin, etc, used during manufacture of biologics are of animal origin. All of these components (substrate, seed stocks, raw materials, etc) pose substantial risks of inherent contaminants and adventitious agents, which may grow during manufacture of the product or grow in the human body after administration of the product. Therefore, all these components require documented history of their origin or isolation and passage history with complete traceability (ie, exposure to various reagents during isolation and propagation). Extensive testing for inherent and adventitious agents, including viruses, mycoplasma, bacteria, yeast, and fungi, and risk analysis for bovine spongiform encephalopathy and transmissible spongiform encephalopathies, are performed on seed stocks, cell banks, batches of media components, etc, at various stages (ie, master and working cell banks or seed stocks, harvests, or other intermediate stages during manufacture). Aseptic manufacturing process seems essential for manufacture of biologics due to the risks discussed above and also due to the fact that biological products being proteins, polysaccharides, carbohydrates, lipids, etc, and growth media (used during manufacture or as a residual component in intermediate components or final product) support microbial growth.
Table 2. Challenges in Achieving Mmicrobiological Quality for Vaccines and Need for Aseptic Processes for Manufacturing Vaccines

Regulation of Biologics and their Microbiological Quality

The challenges in assuring microbiological quality of biologics have been recognized by regulatory agencies around the world for decades, and additional or separate requirements have been in place to regulate biologics.23-26 The US Parts 600 to 680 of 21 Code of Federal Regulations (21 CFR 600–680) describe the regulation of biologics.23 Recognizing the microbial contamination risk during manufacture of biologics, 21 CFR 610.12 specifically required sterility testing on final bulk or Drug Substance (DS) of biologics.27 In practice, biologics, particularly vaccines that are given to millions of healthy babies and infants, sterility tests have been performed at a number of intermediate products, including final bulk, to achieve maximum sterility assurance for vaccine products. In 2012, the sterility test described for biological products in 21 CFR 610.12 was amended to exclude testing at final bulk stage.28 This change could be a significant risk for contamination of vaccines given to healthy individuals and may subsequently lead to adverse reactions in recipients. Until 2012, all biological products had to be sterile from final bulk stage or earlier, usually manufactured aseptically following processes for making sterile drugs. With the amended sterility requirement, the final bulk does not need to be manufactured aseptically and can be manufactured following processes used for making non-sterile drugs. European Pharmacopeia (Ph. Eur.) chapter 7.6 allows replacement of the sterility test for intermediate products with a bioburden test having low limit specifications, with the conditions that intermediate product can be filter-sterilized and the intermediate product does not support microbial growth during storage.29 This requirement can be applied on a case by case basis, based on the risk-benefit ratio and after meeting conditions discussed in Ph. Eur. However, replacing the sterility test for vaccines with a bioburden test at intermediate product and final bulk stages, leading to selection of non-sterile manufacturing processes for vaccines, is not scientifically and technically sound (discussed later). Table 2 summarizes major reasons for employing aseptic processes for manufacturing vaccines. Not employing aseptic manufacturing processes will lead to lack of sterility assurance achieved through EM and aseptic process validation that are not required for the manufacture of non-sterile drugs or components. Further, a bioburden test does not require testing for anaerobic bacteria,5,6 such as Clostridium tetani, Clostridium botulini, etc, which produce lethal toxins. There is a potential risk of contamination with such toxins of products made by non-sterile manufacturing process and not tested for absence of anaerobic bacteria.

General Principles to Control Microbes during Manufacture of Sterile and Non-Sterile Drugs

Building, monitoring, and maintaining cleanroom environment is expensive, and it may not be required or desirable for non-sterile drugs if there is no value for the patient. A careful risk analysis is required to make a decision considering the unique challenges posed by the manufacture of biologics and the use of vaccines in a healthy population as discussed above (Table 2). In contrast to biologics, drugs are usually chemical salts or compounds, often in dry powder form, and do not support growth of microbes during storage, even at room temperature. Therefore, low level of bioburden is usually acceptable, particularly when these drugs are meant for topical, oral, or intranasal use. There is not much risk in using non-sterile processes to manufacture intermediate products or active pharmaceutical ingredients (API) for sterile drugs because these APIs are usually in powder form, and do not support growth of microbes. There are certain general principles to control microbes for manufacture of both sterile and non-sterile drugs.
  • Microbial growth in excipients, APIs, components, and DS should be monitored and controlled to avoid unacceptable levels.
  • Microbial growth is not only a risk for microbial toxins or other toxic components produced during growth, but could also damage the chemical and pharmacological properties of drugs.
  • In particular, microbial proteases could break down proteins in biological products.
  • Manufacturing, testing, and storage facilities should not have any microbial growth, which can be a source of contamination for the raw materials, intermediate products, DS, and DP.
  • Manufacturing and testing facilities should have controlled access with procedures in place to control or prevent entry of microbes in the facility.
  • Lower bioburden levels in DP, components, and raw materials than those required in compendia and product not supporting microbial growth at the recommended storage conditions will control the risk of microbial toxins, and ensure the stability of drugs from microbial degradation.

Aseptic Manufacturing Process and Environmental Monitoring

Table 3. Essential Elements of Aseptic Manufacturing Process
Table 3 summarizes essential elements of aseptic process for the manufacture of sterile drugs. A robust EM program is an essential and critical component to demonstrate the state of control of the facility and the environment required for an aseptic manufacturing process. However, EM is not a direct measure of batch sterility due to inherent variability of methods used to monitor the environment and also due to a lack of a correlation between EM levels and batch sterility.30 Nonetheless, EM provides valuable information about the status of cleanrooms, whether meeting required specifications with regard to particles and viable organisms, the performance of HVAC system, use of acceptable personnel techniques, gowning practices, status of the equipment, and cleaning operations.30 A number of regulatory guidance documents13,14,25 and a recent publication30provide valuable information about the aseptic manufacturing process and requirements for an eff ective and robust EM program. This article is not intended to go into details of all aspects related to aseptic manufacturing. Instead, this article highlights some important aspects that need discussion, particularly aspects important in the manufacture of biologics.


Selection grade or class of cleanroom for each stage of manufacture of biologics is complex and one of the most misunderstood areas in implementing cGMP regulations. A thorough risk assessment approach is an important cGMP tool for an eff ective EM program. Table 4 lists essential components of EM. A basic element of an EM program is the classification of cleanrooms. Currently, there are 3 major systems for the classification of cleanrooms used in the pharmaceutical industry based on the number of air particles >0.5 μ in a cubic foot of air.13-15,25,31 For example, the critical area of aseptic manufacture, Class 100, should not have more than 100 particles of ≥0.5 μm in one cubic foot of air. As per International Standard Organization (ISO) this area is classified as ISO 5,31 which is equivalent to Grade A of European Union’s (EU) GMP guidelines, classified on the basis of metric system (not more than 3520 particles of ≥0.5 μm in one cubic meter of air) and EU grading of cleanrooms is based on counts during operations and at rest.14 Classification of cleanroom is a universal standard, not only for the pharmaceutical industry, but also for other industries (such as electronics) and has been described elsewhere.13-15,25,31 One of the major differences in various regulatory guidance and requirements is the classification of the supporting area for the critical Class 100 area. The FDA guidance document suggests Class 100 in Class 10,000 (ISO 7 or Higher),13 whereas EU GMP requirements and WHO guidelines recommend class 100 in class 1000 or ISO 6.14,25 Class 1000 and class 10,000 areas have significant different specifications for EM parameters, particularly viable organisms. In older vaccine manufacturing facilities, it is sometimes difficult to meet class 1000 specifications for upstream manufacturing processes when there are supporting data on the aseptic process from the purity of a culture during fermentation. The intermediate product is immediately sterile-filtered after confirming purity or bacteriostatic preservatives, inactivating agents, such as formaldehyde are added to detoxify toxins, to inactivate bacteria or viruses or to avoid contamination. Such risk analysis and the impact on the quality of the product will be useful to justify a change in supporting area from Class 1000 to Class 10,000. Use of Class 10,000 supporting area for Class 100 critical area with a risk analysis, as discussed, will be a stringent control than the current regulations of not requiring sterility at the final bulk, leading to classifying the manufacturing process as non-sterile.
Table 4. Major Components of Environmental Monitoring

Environmental Monitoring

Table 4 lists major components of an EM program. It is important to understand these components, which will help in the selection of appropriate methods to implement an effective and robust EM program. Evaluating the quality of air, surfaces, personnel, etc, in a cleanroom environment should start with a well-defined written program employing scientifically sound methods of sampling, testing, data analysis, etc, with an independent oversight by the quality assurance department. Sampling locations and adequate sampling are critical components of an effective EM and should be specified in the written program or standard operating procedures.30 For example, air and surface samples need to be taken at locations with significant activity or product exposure.

Air Monitoring

Air monitoring for total particles is usually done for 0.5- to 5- and >5-μm particles as the cleanrooms are classified based on these counts. Currently, on-line air monitoring systems using remote probes are available to count particles on a continuous basis, both statically and during operations (dynamically). Manual air samplers with well-defined and documented sampling locations, volume of air to be sampled, and sampling frequency may also be used. Sampling locations and placement of probes should be carefully evaluated to collect information that provides status on the quality of the environment during operations. Viable particles (microbes) can be monitored either actively using air samplers or passively by settle plates.30 Historically, microbes have been monitored for aerobic bacteria, yeast, and fungi. Several firms have been using anaerobic incubations of media plates to isolate anaerobic bacteria from cleanroom environments.

Personnel Monitoring

Personnel are the largest risk factor in aseptic manufacturing processes. During each session, gloves and gowns are periodically sampled and monitored for aerobic bacteria, yeast, and fungi with a need to monitor for anaerobic bacteria—particularly Propionibacterium acnes, a facultative anaerobe, which is part of the skin normal flora and has been isolated from manufacturing environments. Personnel health monitoring and medical examination are required for those working in aseptic manufacturing processes. Normal flora from these persons, particularly from nails, hands, hair, etc, may be useful during investigations to find out the source of contamination.

Personnel Training

All operators should be trained and qualified on various procedures, including gowning, with a good understanding of the procedures, their importance in aseptic manufacturing operations, and the impact or risk to quality for not following these procedures. Training on working in a cleanroom should focus on minimizing the generation of particles and disruption of air flow. Examples of personnel training can include aseptic technique, cleanroom behavior, microbiology, hygiene, patient safety hazards due to non-sterile drugs, and specific written procedures on manufacturing operations. For general techniques and operations in cleanrooms, emphasis should be placed on contacting sterile materials with sterile instruments only, with no direct contact of sterile products, containers, closures, or critical surfaces with gown or gloves. In a critical cleanroom area (Class 100), personnel movements should be slow and deliberate in order not to disrupt unidirectional airflow and to avoid turbulence. The entire body should be kept out of the path of unidirectional airflow with a proper gown control.


Samples from surfaces by touch plates or surface swabs are monitored for viable microbes to evaluate the effectiveness of operations, cleaning, and disinfection procedures. Critical surfaces coming in contact with a sterile product should remain sterile throughout an operation.

Analysis of Data and Follow-ups

All EM data should be trended and tracked in real time with the establishment of appropriate alert and action levels based on regulatory guidelines, requirements, and risk-benefit analyses of the product. Averaging the results of EM samples can mask unacceptable conditions.
Investigations for excursions and changes in microbial flora should be thorough with an emphasis on determining the root cause. EM should promptly identify potential root cause of contamination, allowing for implementation of corrections before product contamination occurs.13,30 EM is important to monitor the microbiological quality of critical areas to determine if aseptic conditions are maintained during manufacturing operations.

Selection of Sterile or Non-sterile Manufacturing Process

Sterile drug manufacturing process requires a sterility test at the end to demonstrate the absence of any viable bacterial, yeast, and fungal contaminant. Non-sterile drug manufacturing process requires a bioburden test to provide the number of viable aerobic bacterial, yeast, and fungal organisms that should be lower than the specifications, and absence of objectionable organisms. With the elimination of sterility test at final bulk or DS or replacement of sterility test with the bioburden test, the manufacturing process for that intermediate product, final bulk or DS will be a non-sterile process. This is an important change in the manufacture of biologics, particularly vaccines (Table 2). Lack of much guidance on microbiological controls, no requirements for classification of cleanrooms, and no requirements for EM in manufacture of non-sterile drugs will result in a significant risk to the microbiological quality of vaccines. Can vaccines be manufactured by non-sterile manufacturing process until the final bulk or DS, then filter-sterilized for filling? Answering yes to this question seems scary. This is an example where we need to go ‘back to basics’ and return to the science of applied or pharmaceutical microbiology, as emphasized by Lolas in a recent commentary.32 Based on this author’s experience and knowledge in the manufacture and regulation of vaccines, these products cannot be manufactured like nonsterile drugs and then sterile-filtered at the final bulk before filling. From a historical, technical, and scientific perspective as summarized in Table 2, vaccines have been made under aseptic conditions. Sterile manufacturing process for vaccines will also be supported from a business perspective due to a high risk of microbial contamination during manufacture (Table 2). Microbial contamination will have significant impact on the yield and quality of the final product, leading to rejection of a number of DP lots. Immunization with vaccines has been one of the most successful and cost-effective public health interventions in controlling infectious diseases.33 Changing the manufacturing of vaccines to non-sterile processes has the potential risk of a major public health disaster that will shatter the confidence of the public in safety of vaccines.

Sterility Test and Bioburden Test

As discussed above, a sterility test is required for sterile drugs4 and a bioburden test is required for non-sterile drugs.5,6 Both tests have the limitations of microbiological methods and do not provide absolute results or a complete assurance on the absence of viable organisms. There are some other differences in these tests, which are important to understand, which will provide further rationale that vaccines should not be manufactured by non-sterile processes.
For the sterility test of intermediate products and final bulk, a 10-ml sample is tested in each of 2 media. The amount of sample for a bioburden test depends upon the specification to provide the assurance for that specification. There has been a significant misunderstanding and confusion in setting specifications for a bioburden test and expressing the results. A specification of <1 0.1-ml="" 0.1="" 0="" 1-="" 100="" 10="" 1="" a="" absence="" almost="" an="" and="" any="" are="" as="" assurance="" at="" bacterial="" be="" because="" been="" bioburden="" bulk="" but="" by="" cause="" cfu="" cfus="" colony="" comparing="" considered="" counts="" determine="" discussed="" do="" does="" drugs="" emphasizes="" end="" established="" even="" example="" expressed="" expressing="" expression="" filtration="" final="" for="" forming="" from="" further="" has="" importance="" in="" inaccurate="" intermediate="" interpreted="" is="" lesser="" level="" liquid.="" load="" low="" manufacture="" materials="" may="" microbes.="" microbes="" microbiologists="" microbiology="" million="" misunderstanding="" ml.="" ml="" must="" no="" non-microbiologists="" non-sterile="" not="" of="" often="" on="" or="" particularly="" per="" perspective.="" product="" products="" provide="" raw="" recently.="" regulatory="" required="" respectively="" results="" sample="" scientifically="" specification="" statistically="" sterile.="" sterile="" sterility="" such="" sufficient.="" suitable="" sup="" test="" tested.="" the="" there="" these="" this="" times="" to="" turbidity="" units="" up="" very="" visible="" when="" will="" with="">32
The Ph. Eur. suggests replacing sterility test with a bioburden test for intermediate products during manufacture of vaccines with a low bioburden specification and when product does not support growth of microbes.29A low bioburden specification should be <0 .1="" 10-ml="" 10="" a="" absence="" as="" assurance="" be="" bioburden="" bulk.="" cfu="" despite="" fact="" final="" for="" in="" intermediate="" level="" low="" manufacturing="" ml="" of="" on="" or="" p="" per="" performed="" process="" product="" provide="" require="" sample="" should="" similar="" specification.="" specification="" sterile="" sterility="" still="" such="" test="" testing="" that="" the="" this="" to="" which="" will="" with="">Finally, lack of testing for anaerobic bacteria in the bioburden test is a major limitation and could be a potential risk of contamination of the product with deadly microbial toxins as discussed earlier.


Environmental monitoring and microbiological testing play a critical role in ensuring the safety of patients and the efficacy of drugs and biologics by preventing their contamination with microbes. Microbiological testing alone does not provide complete or absolute assurance of absence of microbial contamination. However, such testing combined with a robust environmental monitoring program and the use of validated manufacturing processes provides a high degree of assurance of the microbial safety of drugs. To build microbiological quality in drugs and biologics, it is important to understand the ways to prevent contamination and risks of microbial growth in intermediate products, components, active pharmaceutical ingredients, final bulk or drug substance, and final product. Manufacturing processes (sterile or non-sterile) should be based on factors such as risk analysis, target population for the drug, and the route of injection.
Rajesh K. Gupta, PhD, is Principal Consultant, Biologics Quality & Regulatory Consultants, LLC, North Potomac, MD 20878, USA


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Potency Testing of Biopharmaceutical Products


Potency determination refers to the quantitative measurement of the biological activity of a given product. Biological activity is a critical quality attribute; therefore, potency testing is an essential component of quality control. Various procedures, including animal-based assays, ligand and receptor binding assays, cell culture-based assays, or other biochemical assays (such as enzymatic assays), may be used for potency testing based on the mechanism of action of the product. This article provides a review of the more commonly adopted assays—specifically ligand and receptor binding and cell-based potency assays, as well as recent advancements in statistical analysis for potency determination and strategies for phase appropriate method development and validation.

Ligand and Receptor Binding Assays

Many biological products, such as monoclonal antibodies, exert their function via binding to a cellular or soluble target, which subsequently triggers appropriate downstream cellular events. For these products, a binding assay offers direct measurement of the product’s affinity to its intended target and may be suitable for potency testing. The most common type of binding assays is the Enzyme Linked Immuno-Sorbent Assay (ELISA), which can be developed relatively quickly and typically offers robust performance. With the advancement of technology, various “homogeneous” immunoassays have been developed and successfully utilized for potency measurement in QC settings. Examples are Time Resolved Homogeneous Fluorescence Resonance Energy Transfer assays, Amplified Luminescence Proximity Homogeneous assays (such as AlphaLISA) and Proximity Based Electrochemiluminescence Immunoassays. These homogeneous immunoassays eliminate the need for wash steps, and the simple “mix and go” procedures result in decreased assay time and potential analyst error. In some cases, superior signal-to-noise ratio and better overall assay performance, as compared to traditional ELISA, may be achieved. However, custom protein conjugation may be required, and assay performance is much dependent on the quality of these critical reagents (tagged proteins, donor and acceptor beads, etc). In addition to immunoassays, Surface Plasmon Resonance (SPR) assays have also been utilized to measure product binding to its intended target. In an SPR assay, protein-protein interaction is detected in real time through changes in mass due to adsorption at the chip surface. Data generated can be used to calculate the binding constant; therefore, SPR assays can be particularly useful during product development. To date, SPR assays have not been used as widely as QC methods for potency measurement but have been adopted sometimes for product characterization, in particular in the field of Biosimilars as part of the comparability study to the innovator products.

Cell-Based Potency Assays

Cell-based potency assays are often the preferred format for potency determination, since they measure the physiological response elicited by the product, which may or may not be extrapolated solely based on demonstration of protein interactions between the product and its intended target. Cell-based potency assays should be developed based on the mechanism of action (MOA) of the product, and therefore, they come in many different formats. The most common types of cell-based assays used to characterize recombinant protein/monoclonal antibodies include proliferation and cytotoxicity assays, apoptosis assays, and assays that measure induction/inhibition of functionally essential signal molecules (such as phosphorylated proteins, enzymes, cytokines and cAMP). Cell proliferation and cytotoxicity assays are essentially cell viability assays. They are most often utilized for products that act through promoting or inhibiting cell growth/killing, such as recombinant growth factors, and Antibody-Drug Conjugate products, which are a common class of cancer therapeutics. Proliferation and cytotoxicity assays typically require prolonged cell culture incubation time and measure viable cell number via quantification of metabolic activity or metabolic substrate (such as ATP). For products that induce cell death via apoptosis pathways, an apoptosis assay measuring the caspase activity offers an alternative, faster method. Activation of the caspase activity is one of the early cellular events that take place in cells undergoing apoptosis. As a result, caspase-based apoptosis assays can often be accomplished within hours, compared to the 2 to 5 days required for traditional cell viability assays. Assays that measure induction/ inhibition of signal molecules tend to be more complex as the quantitation of signal molecules are often accomplished through an ELISA or enzymatic assay. Consequently, both the cell culture treatment as well as the follow up ELISA/enzymatic assay need to be optimized. When a “native” assay poses significant technical challenges that are difficult to overcome, a surrogate assay may be used with sound scientific rationale. For example, reporter gene assays have been frequently used when the intended biological effect has been shown to be mediated through relevant transcriptional regulation events. Reporter gene assays in general offer the advantages of easy set-up, short assay time (1 to 2 days), and reliable assay performance. In addition, once a reporter gene cell line is established, it may be used for the testing of multiple products that have a similar MOA and become a “platform” potency assay. It is of note that recently, Antibody Dependent Cell Cytotoxicity (ADCC) reporter gene assays have been developed and demonstrated with significantly more robust performance than the traditional Peripheral Blood Mononuclear Cells based ADCC assays. The effector reporter gene cell line can be coupled with an appropriate target cell line to assess the ADCC function of any given product. More specialized cell-based potency assays such as phagocytosis assays, cell transduction assays, cell differentiation assays, and viral plaque assays are also employed, whenever appropriate, based on product mechanism of action.

Statistical Analysis in Potency Assays

Many statistical considerations are necessary to support the development of potency assays and to establish suitability for use. In this article, we focus on the concept and implementation of “parallelism testing.” Typically in a potency assay, dose response curves of the test sample and the reference standard are generated, and test sample results are reported as “relative potency” compared to the reference standard. The sample and reference standard dose response curves are compared to determine similarity, or “parallelism.” Only when the dose response curves are parallel, can a meaningful relative potency result be calculated. Historically, classical hypothesis testing (Difference Testing) has been adopted for measuring parallelism. In recent years, there has been a move in the potency testing field towards the “Equivalence Testing” approach. In the new USP bioassay chapters (<1032>, <1033>, and <1034>), theoretical advantages, practical challenges as well as several recommended approaches for implementing the Equivalent Testing are well described. Ideally, the equivalence limit should be set taking into consideration both assay capability and knowledge of product characteristics. Sufficient assay data, generated from the reference standard comparing to itself, to multiple manufacturing lots, and to known “non similar samples” (for example, degraded samples), whenever possible, should be evaluated to determine appropriate acceptance criteria.

Phase Appropriate Potency Assay

Development and Validation Development of a biopharmaceutical product requires significant time and resources and carries a high level of uncertainty. Therefore, it is pragmatic to adopt a phase appropriate strategy for potency method development and validation. During the early stage of development, a binding assay (if appropriate based on MOA) is often preferred over a cell-based potency assay since a binding assay is much easier to develop and implement in a QC environment. However, as the project advances, especially when moving into pivotal clinical trials, a cell-based potency assay is often necessary and is typically preferred by regulatory authorities since it is more physiologically relevant and can sometimes reveal differences in product quality that are not detected in binding assays. It is important to note that some products may have multiple MOAs. In such a case, multiple assays may need to be established to sufficiently demonstrate product efficacy as well as lot-to-lot comparability. As an example, for monoclonal antibodies that are expected to function through direct inhibition of receptor-induced proliferation, as well as Fc function (such as ADCC and Complement Dependent Cytotoxicity [CDC]), a toolbox containing a cell proliferation assay, an ADCC assay, a CDC assay, and an array of chemio-physical assays may be necessary to support both product development and quality control. Once sufficient knowledge has been obtained on product consistency and correlations between results from these different potency assays have been established, it is possible that only one of the assays is selected as a lot release assay to support routine manufacturing campaigns.
Once a potency assay is developed, the sponsor needs to perform a method qualification or validation to demonstrate suitability for intended use. Method qualification/validation also often follows a “phase appropriate” approach. During the early phase of clinical trials, the potency method should at minimum be qualified to demonstrate sufficient accuracy, precision, linearity, and range. The focus on accuracy and precision ensures meaningful interpretation of dose escalation studies. Comprehensive method validation should be implemented as the product moves into Phase III clinical trials and in anticipation of commercialization. A late phase validation study is typically more extensive than that of an early phase qualification and performed under a written protocol that clearly defines the scope of the validation, the target acceptance criteria, and data analysis plan. Multiple analysts and instruments are often employed, and the number of necessary assay runs is justified based on assay variability and intrinsic bias (if known). Method accuracy can be established by testing a sample with known relative potency prepared from the reference standard. In addition, representative routine sample types (drug substance, drug product, etc) should also be tested to confirm suitability of sample handling procedures and method precision. Representative degraded samples—obtained through long term or forced degradation studies—are also frequently included for testing during method validation to confirm the method’s stability-indicating property. Although method robustness may have been established using results generated during method development, a Design of Experiment can often be included within the validation to demonstrate tolerance to varying critical assay conditions.
After successful completion of the method validation, proper assay maintenance should be performed to prevent assay drift. Critical reagents need to be qualified prior to use, and new lots of reference standard need to be calibrated and bridged to the old lot of reference standard. Trending and periodic review of method parameters, such as EC50, signal-to-noise ratio, assay failure rate, as well as relative potency results are also essential components of assay maintenance, especially in a QC environment.

Final Comments

Potency determination is a critical part of product quality control. Potency assays may present in many different formats based on the MOA of the product. Phase appropriate method development and validation strategies help to reduce patient and business risk and are an integral part of product development.