Validation of an index of the quality of review articles.

Abstract

The objective of this study was to assess the validity of an index of the scientific quality of research overviews, the Overview Quality Assessment Questionnaire (OQAQ). Thirty-six published review articles were assessed by 9 judges using the OQAQ. Authors reports of what they had done were compared to OQAQ ratings. The sensibility of the OQAQ was assessed using a 13 item questionnaire. Seven a priori hypotheses were used to assess construct validity. The review articles were drawn from three sampling frames: articles highly rated by criteria external to the study, meta-analyses, and a broad spectrum of medical journals. Three categories of judges were used to assess the articles: research assistants, clinicians with research training and experts in research methodology, with 3 judges in each category. The sensibility of the index was assessed by 15 randomly selected faculty members of the Department of Clinical Epidemiology and Biostatistics at McMaster. Authors' reports of their methods related closely to ratings from corresponding OQAQ items: for each criterion, the mean score was significantly higher for articles for which the authors responses indicated that they had used more rigorous methods. For 10 of the 13 questions used to assess sensibility the mean rating was 5 or greater, indicating general satisfaction with the instrument. The primary shortcoming noted was the need for judgement in applying the index. Six of the 7 hypotheses used to test construct validity held true. The OQAQ is a valid measure of the quality of research overviews.

Oxman AD1, Guyatt GH.

Author information

• 1Department of Family Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada.

Applying Continuous-Flow Pasteurization and Sterilization Processes

Photo courtesy of MicroThermics, Inc.

High-temperature, short-time (HTST) pasteurization and ultra-high temperature (UHT) sterilization are continuous-flow thermal processes that have been established and highly refined in other industries for many years. Their precision and minimal impact enable the manufacture of products that cannot be made using batch technologies. HTST and UHT are traditionally used for heat-sensitive products. As continuous-flow processes, they are effective against vegetative cells, viruses, and heat-stable endospores. These characteristics and their continuous-flow nature make them potentially useful as part of the trend toward methods of continuous manufacturing of bio/pharmaceuticals. Technologies frequently evolve separately, often independently, in unrelated industries and transfer between them. This transfer is often how industries and technologies advance and take significant steps forward. Identifying a technology that is proven, highly refined, and fully supported industrially and regulatorily, however, is less common. This is the case for high-temperature, short-time (HTST) pasteurization and ultra-high temperature (UHT) sterilization. HTST and UHT are continuous flow, thermal processes that have been used to pasteurize and sterilize liquids (e.g., foods) for more than 60 years. The processes have been developed as tightly controlled systems and refined to reliably produce high quality products at low cost. HTST and UHT have been optimized to reach high assurance levels for inactivation of vegetative cells, viruses, and heat-stable endospores, all while retaining quality that could not be maintained using batch processes, such as autoclaving.
HTST and UHT in the Food Industry
It seems somehow concerning to use a process that has evolved for food products, such as milk and juices, and use it for highly refined pharmaceuticals, but let's consider the fundamentals. The chemical-reaction kinetics that describe how and why these processes inactivate bacteria but retain the quality in a biological fluid (e.g., milk) are the same as in any other biological fluid. Commercially, these processes are well established and used for products ranging from juices to baby food and even products as sensitive as liquid whole egg. These processes have annual capacities measured in hundreds of billions of packages per year. Commercial equipment for HTST and UHT processes commonly operate at flow rates ranging from roughly 5 gallons to more than 100 gallons per minute. Commercial capacities, however, do not lend themselves to the batch sizes and rapid cycles of research and development. The need to conduct thorough research and to optimize treatments (e.g., hold time, temperature, and heat transfer) for different products has triggered the development of miniaturized research equipment and experimental methods for this purpose. These tools have enabled R&D professionals to address potential manufacturing issues early and avoid losses and costly problems while also helping these processes to become better understood. Optimization of these processes has led to development of a wide assortment of time and temperature treatments as well as highly refined tools to test products and deliver these treatments. As a consequence, small-capacity systems have been developed for lower flow rates, bringing the benefits of HTST pasteurization and UHT sterilization to the high-value, low-volume materials of pharmaceuticals.  Sterliization Continuous Manufacturing
Continuous manufacturing has been described as a manufacturing breakthrough and as the method of the future by Konstatine Konstantinov, vice-president of commercial process development at Genzyme (now part of Sanofi), and Robert Bradway, chairman and CEO of Amgen (1, 2). The trend toward using this method is increasing as manufacturers of bio/pharmaceuticals strive to meet growing demand, reduce floor space, improve manufacturing flexibility and capacity, and reduce costs.
The adoption of continuous manufacturing for biopharmaceuticals emphasizes the need to inactivate microorganisms continuously at rates consistent with these new processes. The adoption of HTST and UHT continuous processing and surrounding technologies is a natural fit. Early adopters in the biotechnology and biopharmaceutical industries have begun to deploy these processes. The question remains, however, what are the reasons to adopt HTST and UHT in these industries? Are their benefits simply a function of the continuous process or are there additional benefits that make HTST and UHT even more desirable?

Figure 1: Flow diagram for continuous-flow thermal processes. (ALL FIGURES COURTESY OF AUTHOR)

Benefits ofF HTST and UHT
The benefits of HTST and UHT processes result from their continuous flow nature and their use of different and more highly refined time and temperature conditions. To understand their benefits, it is useful to consider an example process like that shown in Figure 1. The product is pumped continuously through the process at constant flow and is heated to the process temperature under steady-state conditions. It flows through the hold tube, which is of sufficient length to ensure that the product is hot for the time needed for the required lethality, before it is cooled as it exits the system. The result is that the product experiences a controlled, well-defined time–temperature exposure. This time–temperature history (TTH), conceptually shown in Figure 2, is usually less than two minutes from start to finish. Although there are relatively few rules linking the terms "pasteurization" or "sterilization" to specific temperatures, for the sake of this discussion, pasteurization is usually conducted at hold-tube temperatures between 70 °C and 121 °C. Sterilization hold temperatures range from 128 °C to 150 °C. Hold times most commonly range from 2 to 30 seconds.

Figure 2: Conceptual plot of product time–temperature history.

Because HTST and UHT continuous-flow processes are closed systems that operate at steady state, the impact of the thermal process on product quality and lethality is uniform and independent of batch and container size. In contrast, the thermal exposures delivered to liquids being pasteurized or sterilized in large-scale fermenters or vessels in autoclaves/retort are not as uniform because the heat exposure varies with the container size and location. In comparison, it becomes apparent that, in the continuous process, several major sources of variation and potential points of failure have been eliminated and overprocessing has been greatly reduced. As thermal processes, HTST and UHT processes are effective against viral contamination. These processes are especially useful for emulsions and suspensions that are not compatible with filtration. They provide real-time monitoring and record-keeping of processing conditions. They are precise, and the actual process time and temperature conditions can be adjusted to optimize the retention of key components and the delivered lethality. This precision can be important to maximize retention of key media components being fed into a fermentation process or of a desired active agent resulting from a different step of manufacture.
Unlike scale-up of batch operations, scale-up of HTST and UHT processes is often unnecessary because processing more material is linked only to the processing time, not the vessel size. Larger volumes of product are processed by simply running the equipment longer. Thus, multiple systems may not be needed for different batch sizes. When scale-up is necessary within the same general style of HTST or UHT equipment, it is a matter of duplicating the TTH. If a different style system is used, the detailed matching of the TTH may require more powerful mathematical and modeling tools for thermal process evaluation.
In the food industry, these processes are used to make many high quality products that would not be viable using longer-time and lower-temperature methods, such as autoclaving, because of poor quality. These examples demonstrate the potential to pasteurize or sterilize many bio/pharmaceutical materials that are also not well-suited to autoclaving. In simpler terms, these are enabling technologies. 
Sterliization Continuous Manufacturing
Continuous manufacturing has been described as a manufacturing breakthrough and as the method of the future by Konstatine Konstantinov, vice-president of commercial process development at Genzyme (now part of Sanofi), and Robert Bradway, chairman and CEO of Amgen (1, 2). The trend toward using this method is increasing as manufacturers of bio/pharmaceuticals strive to meet growing demand, reduce floor space, improve manufacturing flexibility and capacity, and reduce costs.
The adoption of continuous manufacturing for biopharmaceuticals emphasizes the need to inactivate microorganisms continuously at rates consistent with these new processes. The adoption of HTST and UHT continuous processing and surrounding technologies is a natural fit. Early adopters in the biotechnology and biopharmaceutical industries have begun to deploy these processes. The question remains, however, what are the reasons to adopt HTST and UHT in these industries? Are their benefits simply a function of the continuous process or are there additional benefits that make HTST and UHT even more desirable?

Figure 1: Flow diagram for continuous-flow thermal processes. (ALL FIGURES COURTESY OF AUTHOR)

Benefits ofF HTST and UHT
The benefits of HTST and UHT processes result from their continuous flow nature and their use of different and more highly refined time and temperature conditions. To understand their benefits, it is useful to consider an example process like that shown in Figure 1. The product is pumped continuously through the process at constant flow and is heated to the process temperature under steady-state conditions. It flows through the hold tube, which is of sufficient length to ensure that the product is hot for the time needed for the required lethality, before it is cooled as it exits the system. The result is that the product experiences a controlled, well-defined time–temperature exposure. This time–temperature history (TTH), conceptually shown in Figure 2, is usually less than two minutes from start to finish. Although there are relatively few rules linking the terms "pasteurization" or "sterilization" to specific temperatures, for the sake of this discussion, pasteurization is usually conducted at hold-tube temperatures between 70 °C and 121 °C. Sterilization hold temperatures range from 128 °C to 150 °C. Hold times most commonly range from 2 to 30 seconds.

Figure 2: Conceptual plot of product time–temperature history.

Because HTST and UHT continuous-flow processes are closed systems that operate at steady state, the impact of the thermal process on product quality and lethality is uniform and independent of batch and container size. In contrast, the thermal exposures delivered to liquids being pasteurized or sterilized in large-scale fermenters or vessels in autoclaves/retort are not as uniform because the heat exposure varies with the container size and location. In comparison, it becomes apparent that, in the continuous process, several major sources of variation and potential points of failure have been eliminated and overprocessing has been greatly reduced. As thermal processes, HTST and UHT processes are effective against viral contamination. These processes are especially useful for emulsions and suspensions that are not compatible with filtration. They provide real-time monitoring and record-keeping of processing conditions. They are precise, and the actual process time and temperature conditions can be adjusted to optimize the retention of key components and the delivered lethality. This precision can be important to maximize retention of key media components being fed into a fermentation process or of a desired active agent resulting from a different step of manufacture.
Unlike scale-up of batch operations, scale-up of HTST and UHT processes is often unnecessary because processing more material is linked only to the processing time, not the vessel size. Larger volumes of product are processed by simply running the equipment longer. Thus, multiple systems may not be needed for different batch sizes. When scale-up is necessary within the same general style of HTST or UHT equipment, it is a matter of duplicating the TTH. If a different style system is used, the detailed matching of the TTH may require more powerful mathematical and modeling tools for thermal process evaluation.
In the food industry, these processes are used to make many high quality products that would not be viable using longer-time and lower-temperature methods, such as autoclaving, because of poor quality. These examples demonstrate the potential to pasteurize or sterilize many bio/pharmaceutical materials that are also not well-suited to autoclaving. In simpler terms, these are enabling technologies. 
The Learning Curve in Bio/Pharmaceuticals
The technologies of HTST pasteurization and UHT sterilization have been refined for many years in other industries. Details of validation, maintenance of proper safety assurance, and documentation have also been well defined. The tools to generate thermal-destruction kinetics, test products, and optimize operating conditions simplify the adoption of these technologies for bio/pharmaceutical manufacture.
These technologies have been developing for many years, and their adoption involves the use of the existing tools to optimize them for each application. It also requires specific efforts to implement them properly. As continuous-flow processes that are new to a manufacturing environment, they require training and a different outlook to support this transition. It is a cultural change, which extends from manufacturing through maintenance, quality assurance, engineering, and management.
Experience in other industries has shown that thorough initial testing of products is important. Screening to determine the suitability of materials and selecting processing conditions are significant initial steps. Test-processing products thoroughly is essential to obtain data supporting optimization of these conditions. It is equally essential that testing demonstrates the performance of the product with down-line unit operations, especially in continuous manufacturing.

Industry participants are finding that HTST pasteurization and UHT sterilization provide another tool for the manufacture of bio/pharmaceuticals. They have found products for which theses processes are strikingly successful and those for which they are not suitable. More importantly, perhaps the most interesting benefit is the ability to facilitate the development of entirely new products. John Miles, PhD, is president of MicroThermics Inc., 3216-B Wellington Ct., Raleigh NC 27615, tel. 919.878.3777, jmiles@microthermics.com
REFERENCES
1. K. Weintraub, "Biotech Firms Race For Manufacturing Breakthrough," MIT Technology Review Business Report, Jan. 30, 2013, http://www.technologyreview.com/news/509336/biotech-firms-in-race-for-manufacturing-breakthrough/.
2. A. Jungbauer, Biotechnol. J. 6 (12), 1431–1434 (2011).

TOC is effective for cleaning validation.

The total organic carbon (TOC) test is a fast and effective analytical technique to evaluate the cleaning of biopharmaceutical manufacturing equipment. This technique can help ensure that the cleaning processes meet predetermined cleanability criteria for single and multiproduct production areas. This article presents a case study describing the use of the TOC test to validate the cleaning processes used for two types of biomanufacturing equipment.

Baxter Healthcare

Anumber of US and European documents describe the requirements and guidelines for biomanufacturing equipment cleaning processes.¹ Also, many journal articles have discussed strategies for performing cleaning validation.^2–5 However, cleaning validation problems, including a lack of documented procedures, inadequate training of operators, and insufficient validation of analytical or cleaning methods, still are among the four most commonly cited problems in Form 483s and warning letters issued by the US FDA.^6,7 This article discusses a strategy to validate biopharmaceutical facility cleaning processes using the total organic carbon method (TOC). An initial TOC measurement system qualification was performed, followed by an evaluation of the correlation between TOC and microorganism levels. Later, a swabbing recovery study was carried out with cells and proteins. Finally, the cleaning process for two types of biomanufacturing equipment was validated using the TOC test.
MATERIALS AND METHODS
MATERIALS Biological Samples
The cells and proteins used in the swabbing recovery study included Escherichia coli bacterial cells, Saccharomyces cerevisiae yeast cells, recombinant streptokinase (SK), recombinant epidermal growth factor (EGF), recombinant human alpha interferon (IFNα), and pegylated recombinant human alpha interferon (IFNα–PEG).
TOC Vials and Swabs
Glass 40-mL autosampler vials with caps were used for TOC measurements. For the recovery study a TX3340 TOC cleaning validation kit, containing 12 Eagle Picher 03464 40-mL clear vials, 24 Texwipe TX714K large SnapSwabs, and 12 blank vial labels was used. The 40-mL vials were certified as having TOC levels <10 and="" at="" certified="" levels="" of="" ppb.="" ppb="" span="" swabs="" the="" toc="" were="">
METHODS TOC Measurement Method Validation
The TOC test involves full oxidation of organic carbon and detection of the resulting CO₂. In this study, a TOC analyzer equipped with an auto-sampler was used. The analyzer measures TOC according to ASTM method D6317.⁸ It determines the amount of total carbon (TC), inorganic carbon (IC), and TOC in water in the range of 10 to 1,000 μg/L. The test method used persulfate and ultraviolet (UV) oxidation of organic carbon, coupled with a CO₂ selective membrane to recover the CO₂ in deionized water. The change in conductivity of the deionized water was measured and compared with the carbon concentration in the oxidized sample. IC was determined in a similar manner, but without the oxidation step. In both cases, the sample was acidified to facilitate CO₂ recovery through the membrane.
The relationship between the conductivity measurement and carbon concentration is described by a set of chemometric equations for the chemical equilibrium of CO₂, HCO₃–, and H+, and for the relationship between the ionic concentrations and conductivity. The chemometric model includes the temperature dependence of the equilibrium constants and the specific conductance, resulting in a linear response of the method over the stated range of TOC.

The TOC measurement method was validated according to the ICH Q2 (R1) guideline.⁹ Precision and accuracy were calculated using the TOC sucrose standard of TOC. For precision, the standard deviation (SD), and relative standard deviation (RSD) for the three TOC concentrations (250, 500, and 750 ppb; with three replicates for each concentration) readings were determined as follows: in which Σ is the sum of each result and n is the number of measurements in a set (number of replicates – number of rejections). The RSD = (SD/measured TOC concentration) x 100. The accuracy (percent difference between both the expected and measured TOC value) was calculated as follows:
% difference = (measured concentration – expected standard concentration/expected standard concentration) x 100.

Method linearity was verified using the sucrose standard at three concentrations, 250, 500, and 750 ppb of TOC (for each concentration with three replicates). The correlation coefficient (R²) was calculated as follows: in which X is the certified values of TOC standards and Y is the measured values of TOC standards (blank–corrected).
The limits of detection and quantitation (LOD and LOQ, respectively) were calculated using three sucrose standards at 250, 500, and 750 ppb of TOC (for each concentration with three replicates). Using a least squares linear regression algorithm, the equation of the line relating the SDs of the standards to the actual measured concentrations of the standards was determined. The y-intercept represents the SD at zero ppb of standard sucrose concentration. The LOD was the yintercept value multiplied by 3, and the LOQ was the y-intercept value multiplied by 10.
Sample Preparation for Determining the Correlation Between TOC and Microorganisms

Figure 1

Both bacterial and yeast broths were grown until an optical density of 0.6 was reached, at 500 nm and 600 nm, respectively. The culture media used to grow bacteria was LB (5 g/L yeast extract, 10 g/L triptone, 10 g/L NaCl). The culture media used for yeast was YPD (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract). These cell suspensions were centrifuged in a TJ-6 centrifuge (Beckman Instrument, Fullerton, CA) for 10 min at 1,000g. The pellets were washed three times and suspended with low TOC purified water. The serial dilutions for wet cell concentrations shown in Figure 1 also were prepared using the same purified water. Swab Recovery
The coupons used in this work were representative of the materials of construction of the manufacturing equipment.
Therefore, both 100 cm² stainless steel (316 L) and 100 cm² borosilicate coupons were used in the swab recovery study. One side of each coupon was spiked with a solution (or biologic sample) of cells or pure recombinant proteins, respectively. For bacterial and yeast cells, 200 μg, were applied. In the case of recombinant proteins, 1.2 mg of SK, 100 μg of EGF, 100 μg of IFNα, and 12.5 mg of IFNα–PEG, were applied. The coupons were allowed to dry completely for 4 h at 25 °C. A swab was moistened with low TOC water and the spiked coupon surfaces were swabbed both vertically and horizontally. The swab end was cut off and placed in a vial with 40-mL of TOC water. The vials were capped, shaked vigorously by inversion, and the TOC analysis was performed. The same quantity of each analyzed biologic sample was spiked directly in 40-mL of TOC water (reference control).
Manufacturing Equipment Cleaning
The cleaning process was performed using sodium hydroxide and orthophosphoric acid, as described by Verghese¹⁰ and Mollah,⁵ following the standard procedures previously developed by the manufacturing department and approved by quality assurance staff of the Center for Genetic Engineering and Biotechnology (CIGB).^11–13 The final rinses of the fermentation and chromatography equipment were performed with purified water and water for injection (WFI), respectively.
RESULTS AND DISCUSSION
TOC Measurement System Performance
According to the European good manufacturing practice (GMP) regulations, analytical methods used in biomanufacturing equipment cleaning should be able to detect the established acceptable level of the residues or contaminant.¹⁴ Also, according to US FDA cGMP regulations and the USP, the suitability of all compendial testing methods used must be verified under normal laboratory conditions.^15–16
Per the above regulations and considering that this is a quantitative test for impurities, the analytical parameters including accuracy, precision, linearity, detection limits, and quantiation limits, were evaluated with their corresponding assumption, per ICH Q2 (R1).⁹

Table 1. Validated analytical parameters and the key results obtained

The results obtained for each of the above parameters during the TOC analysis are summarized in Table 1. In the case of precision, 1.27% of RSD corresponds to 7.21 ppb of TOC. The linearity showed a fit correlation between ppb of TOC and the conductivity signal from its full oxidation. The lowest amount of TOC quantified in 21 ppb (21 ng/mL) demonstrated high sensitivity of this measurement system. In addition, the suitability test was performed according to the procedure and specifications described in section 643 of the USP31–NF26 and 2.2.44 of the European Pharmacopeia (EP, data not shown).^16–17
Correlation Between TOC and Microorganisms
The bacterial and yeast cells commonly used in fermentation represent the "worst case scenario" in manufacturing system cleaning, because they lead to organic contaminants such as nucleic acids, lipids, carbohydrates, proteins, and endotoxins. The TOC test should provide a linear correlation between the measured molecules and the TOC response.
To analyze the relationship between cell concentration and TOC response, a correlation analysis was performed (Figure 1). In both cases, correlation coefficients were >0.983, showing linear relationship between wet cell concentration and the TOC values. Therefore, contamination with cells or any of those components can be quantitatively determined using the TOC measurement in a wide dilution range. These data can be used to extrapolate the potential wet cell concentration with its corresponding cell number estimate or relative organic carbon contribution in the final results of any cleaning process of a bioprocess equipment.¹⁸
Considering that both bacterial and yeast cultures were harvested at an exponential growth phase, we assumed that the majority of the cells were viable, although some lysed cells were not discarded.
It is significant to note that in first 27 ppb (27 ng/mL) of TOC, one could have a wet bacterial cell concentration of 1.29 mg/mL, which is equivalent to 10⁶ E. coli cells. Similarly, in as little as 16 ppb (16 ng/mL) of TOC, one could have a wet yeast cell concentration of 0.826 mg/mL, which is equivalent to around 10³ yeast cells.
However, according to the WFI specification (500 ppb of TOC) indicated in EP16 and the USP17, the TOC values (27 and 16 ppb) are more than one order below the specification. Thus, the amount of residue found in a final wash sample from manufacturing equipment cleaning could be considered as the "acceptable level" of undesirable residues. To use this value as acceptable level, the TOC value of the water used for cleaning the manufacturing system must be taken as the cleanliness reference.
Swabbing Recovery Study for Cells and Proteins
There are two types of sampling done following the cleaning process—the direct surface and rinse samples. The first one is considered "the most desirable" by the FDA.¹ The swab technique typically involves moistening a polyester swab with purified water to wipe a measured area in a systematic manner. Cleaning validation kits, specifically designed for TOC swabbing, are commercially available for this purpose. However, it is critical to validate the swabbing procedure in combination with the chosen analytical method for various combinations of contaminants and surfaces. Thus, TOC recovery study is mandatory before evaluating the efficiency of the cleaning process. In the absence of such a validation study, a manufacturer may erroneously assume that the equipment is clean, based on an apparent negative result.

Table 2. Values (%) of the cells and recombinant proteins recovered in the recovery study (SD: standard deviation; CV: coefficient of variation)

To evaluate the efficiency of swabbing, the "worst case scenario" was evaluated using fermentation and purification facilities as examples. Moreover, three recombinant proteins were included in this study: SK and interferona 2b human recombinant (IFNα2bHu-r) were obtained from bacterial cells. The EGF was expressed in yeast cells. In all cases, the studies were performed considering the interaction of these cells with different manufacturing processing surfaces, which may in turn significantly affect the swabbing recovery during cleaning validation. The results obtained from microbial host and recombinant proteins on stainless steel and borosilicate glass are shown in Table 2. The mean of recovery factor ranged from 89 to 108%. For the cells, the results are closest to 100% recovery from both surfaces. These results are superior to those published in a similar study by using bacterial whole cell homogenate and stainless steel coupons.¹⁸ Those studies obtained recovery values of 67%, using 0.05 N phosphoric acid as the extraction solution. In a similar study, a recovery mean of 81.9% was obtained using 1 N sodium hydroxide as the solvent using E. coli and stainless steel coupons.² For the recombinant proteins, the lowest recovery value was obtained with SK on stainless steel coupons with a general recovery factor of 89.47%. The remaining recombinant protein–surfaces combinations showed recovery factor values ranging from 93.89 to 107.86%. These results match with those obtained by Lombardo, et al. in a similar study, but using purified recombinant human ciliary neurotrophic factor (rHCNTF) on a stainless steel surface and 0.05 N phosphoric acid as the extraction solution.¹⁸ However, Lombardo's results from borosilicate analysis (55% of recovery factor) were lower than those obtained in our study (from ~94 to 108%).
These results suggest that purified water with low levels of TOC can be used efficiently to recover all biological materials. In addition, the almost identical recovery factor values for the IFNα2bHu-r–PEG and IFNα2bHu-r show that the polyethylene glycol conjugation process does not affect the electrostatic interaction with the borosilicate surface and can be removed efficiently under the conditions described (see materials and methods above).
CLEANING VALIDATION OF TWO TYPES OF EQUIPMENT
Considering the results shown above, the TOC test was used to assess the cleaning of bioproduction equipment used in two types of manufacturing processes. The first set of equipment was used to obtain a recombinant protein from E. coli bacterial cells as host, and in the second scenario, S. cerevisiae yeast cells are used to express a heterologous protein. In both manufacturing systems, one fermentation vessel and one chromatography column were taken as examples.
Before the cleaning process was performed, the procedures used (who is responsible for performing and approving them, acceptance criteria, revalidation) were documented in detail.¹ Then, the cleaning process was performed, as described in preapproved protocols.^11–13

Table 3. Cleaning validation results (TOC, in ppb) for fermenters used in E. coli and S. cerevisiæ production processes

The results of three independent cleaning processes for both manufacturing systems are shown in Tables 3 and 4. For the fermenter vessel, cleaning validation results from rinse samples showed a reduction factor of three orders of magnitude, with final values ranging from 1.27 x 10³ to 2.25 x 10³. The values from the harvest port swabbing samples showed the same trend as for rinse samples, with a three orders of magnitude, and final values ranging from 1 x 10³ to 3.73 x 10³ . On the other hand, the results of chromatography column cleaning showed, in general, lower reduction factor values compared with those for fermenter cleaning. Those results were expected, knowing that the chromatography system is in contact with cleaner material than that seen in fermentation steps.
The final samples (rinse and swabbing samples) of both manufacturing systems showed TOC values as low as 22 ppb (close to the LOQ, Table 1). The TOC value obtained for the water used to clean the biomanufacturing system was used as a reference value.

Table 4. Cleaning validation results (TOC, in ppb) for chromatography columns used in E. coli and S. cerevisiæ production processes

The results of the swabbing recovery study (Table 2) showed that the levels of organic charge reduction after cleaning reported in this work, showed a high level of cleaning efficiency. This proposed cleaning validation strategy can be applied to both single and multiproduct manufacturing facilities. CONCLUSIONS
A general strategy for assessing cleaning validation using the total organic carbon (TOC) test was applied. The qualification of the TOC measurement system showed a precision of 1.27%, 1.3% of accuracy, and a detection limit of 6.30 ppb of TOC.
A linear correlation between cells and TOC values was demonstrated with this analytical system. This study showed that in both 27 ppb and 16 ppb of TOC, ~10⁶ E. coli cells and 10³ S. cerevisiae cells were present, respectively. The swab recovery assessment showed recovery factor values ranging from 90 to 110% using stainless steel and borosilicate surfaces and SK, EGF, IFNα2bHu-r, as well as both E. coli cells and S. cerevisiae as biological matrices, and purified water as a recovery solvent. It also showed that applying the polyethylene glycol conjugation process to IFNα2bHu-r does not affect its interaction with borosilicate surfaces. Given the cleaning reduction factors values, the final TOC values for the studied systems, the agreement between them, and the TOC values from final samples and for the water used to clean each system, the strategy described here can be a useful tool in similar cleanliness validation strategies.
ACKNOWLEDGMENTS
The authors want to thank Magaly García Blanco for proofreading this manuscript and the epidermal growth factor and interferon manufacturing personnel from the CIGB for supplying the cleaning validation samples used in this work.
Julio César Sánchez is the head, and Raudel Sosa and Meily Sánchez are technicians in the process control department, Rebeca Bouyon Albarran is a research scientist in the epidermal growth factor department, Luciano Hernández and Marbel Ramos Alfonso are research scientists in the interferon manufacturing department, Alexis Musacchio Lasa is a research scientist in the bioinformatics department, and Leopoldo Núñez is a technical service assistant, all at the Center for Genetic Engineering and Biotechnology, Havana, Cuba, +537.271.6022 (ext. 7101), julio.sanchez@cigb.edu.cu

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8. ASTM international standard. Available from: http://www.astm.org/Standards/D6317.htm|~http://www.astm.org/Standards/D6317.htm .
9. ICH Q2 (R1) Validation of analytical procedures: Text and methodology. Available from: www.ich.org/LOB/media/MEDIA417.pdf.
10. George Verghese. Cleaning agents for biopharma manufacturing. Gen Eng News. 2003;23(6):46–52.
11. IT 60.602 Preparation and application of API to chromatographic systems for cleaning validation in campaign changes.
12. IT 60.201 Preparation and application to Fermentors of biomass used in the cleaning validation in campaign changes.
13. PVL-005 Procedure of parenteral Epidermal Growth Factor manufacturing equipment cleaning validation.
14. Working Party on Control of medicine and inspections. Qualification and validation. Annex 15 to the EU guide to good manufacturing practice. European commission: Brussels, Belgium, July 2001. Available from: http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-4/pdfs-en/v4an15.pdf|~http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-4/pdfs-en/v4an15.pdf .
15. US Food and Drug Administration. Current good manufacturing practice regulations; 21 CFR 211.194 (a) (2), 2005.
16. United States Pharmacopeia. USP 31–NF 26, USP <643> Total Organic Carbon. 2007 Nov.
17. European Pharmacopeia Method 2.2.44 – total organic carbon in water for pharmaceutical use, European Pharmacopoeia Commission; 6th ed, 2009.
18. Lombard S, Inampudi P, Scotton A, Ruezinsky G, Rupp R, Nigam S. Development of surface swabbing procedures for a cleaning validation program in a biopharmaceutical manufacturing facility. Biotechnol Bioeng. 1995;48:513–519.

Why the Swab Matters in Cleaning Validation

In recent years, increased emphasis
has been placed on the development of
validated and robust cleaning protocols.

WHAT IS CLEANING VALIDATION?

The U.S. Food and Drug Administration (FDA) issued its Guide to Inspections—Validation of Cleaning Process¹ in 1993. Since that time, the protocols surrounding cleaning processes in pharmaceutical manufacturing environments and sampling and filling suites have received increased attention.^2,3 The primary regulatory concern driving the need for cleaning validation is cross contamination of the desired drug substance either by other active pharmaceutical ingredients (API) from previous batch runs or by residues from the cleaning agents used.
Cross-contamination with extraneous residues of any kind presents a safety risk to patients consuming the drug product. It threatens to alter the strength, chemical identity, and integrity of the drug substance and formulation. Therefore, the equipment and work environments involved in drug manufacturing processes must be cleaned at regular, prescribed intervals to prevent the possibility of such cross-contamination. These cleaning protocols must be validated in order to provide assurance that they do, in fact, serve their purpose—to clean the surfaces to a level that avoids the possibility of cross-contamination.
In recent years, increased emphasis has been placed on the development of validated and robust cleaning protocols given the concerns over the safety of our drug supply. Growth in the levels of outsourcing and off-shoring of pharmaceutical manufacturing has heightened the FDA’s concern over cleaning processes. Inadequate documentation, training, and validation of cleaning processes rank high among the four most often cited problems in Form 483 and warning letters that have been issued by the U.S. FDA.⁴

WHY SWABBING?

In a typical pharmaceutical manufacturing environment, cleaning might be performed by using 70% isopropyl alcohol (IPA) and/or other chemicals, detergents, and sanitizing agents in order to remove residues from the previous batch run. The areas thus cleaned must now be sampled adequately and appropriately in order to validate the cleaning protocol.
Swabbing and rinsing are the two most common techniques used for sampling of such cleaned surfaces. Swabbing is a direct surface sampling method, while rinsing is an indirect method. In practice, physical access to surfaces and parts of equipment to be cleaned tends to drive the choice of sampling method. For example, swabbing would work particularly well in more restricted work areas such as isolators, hoods, and accessible corners of equipment, while rinsing would work best in pipes and longer tubes. In general, a combination of both is most desirable in order to accomplish the most comprehensive coverage of surfaces to be cleaned.
While the FDA guidance indicates a preference for the more direct swabbing method, more recent communication from the International Conference on Harmonisation (ICH) ICH Q7A5 states that sampling methods need to be comprehensive enough to quantitate both soluble and insoluble residues that are left behind on the surfaces after cleaning. The exact protocols prescribed will necessarily vary depending on the nature of the products, residues, and surfaces. These protocols must be tailored to the needs of each environment.

THE SWABBING PROCEDURE – CONSIDERATIONS

The swab to be used for sampling is typically pre-wetted with water or another appropriate solvent in order to remove residues from the surface. Squeezing the sides of the swab against the inside of the vial upon pre-wetting prior to sampling removes excess solvent.
This is important because excess solvent can itself serve as a source of residues leading to variable results. There is a direct, physical interaction between the swab, the solvent, and the residues to be removed; therefore, the choice of swab is critical to the effectiveness of the sampling process. The swab used must offer ultra-low particulates and fibers, high absorbency, and minimal extractable interferences. Polyester swabs are specially processed to meet the stringent requirements associated with cleaning validation protocols.
The physical nature of the swabbing process implies that significant levels of operator training be conducted prior to implementation of cleaning validation protocols. This training should serve to minimize the subjectivity that is inherent to such sampling activity. The recommended directions and motions used in actual swabbing of an area as shown in Figure 1 should be detailed in the training to ensure the highest levels of consistency. Alternate swab sampling patterns may certainly be used if they would help maximize percent recovery.
A suitable extractable solvent is used to release the residues from the swab head. Depending on the particular SOP in each area, this swab sample may need to be filtered and/or sonicated to extract the residues as completely as possible. These sample prep procedures place a heavy premium on the intrinsic quality of the materials used in the swab head and the filters. The use of anything less than the highest quality of suitably pre-treated polyester swabs can prove to be a source of extraneous contamination in the subsequent assay.
The method development and validation steps are often conducted on test coupons to serve as examples of the equipment or surfaces to be cleaned. The choice of filter and solvent used in sample preparation is also critical since they can have an impact on the recovery, influence extractables, and efficiency of filtration. Yang et al. have reported a systematic study of a variety of solvent conditions and pH and their impact on the percent recovery and efficiency of filtration.6  While it may be intuitive to choose the solvent conditions used in the subsequent analysis (e.g. HPLC) as the extractable solvent, this may sometimes compromise the filtering efficiency and the percent recovery.

Figure 1: Recommended directions and motions of swabbing.

ANALYSIS OF RESIDUES – ANALYTICAL CONSIDERATIONS

The purpose of swab sampling as part of a cleaning validation protocol is to be able to prove that the cleaning process served its purpose. That purpose (cleaning the surfaces to avoid any cross-contamination) is best measured in the validation step as a percent recovery of seeded residue. Such a measurement provides an estimate of Residue Acceptable Limit (RAL). The measurement of percent recovery is accomplished through an analytical test, typically either HPLC (High Performance Liquid Chromatography) or TOC (Total Organic Carbon).
HPLC-UV systems commonly carry additional detectors such as mass spectrometry (MS - for specificity and identification). It is important to realize early in the method development process for cleaning validation that percent recovery will be directly influenced by the interaction of the particular assay detector with each of the variables involved in the protocol. It is best to conduct a pre-study of the influence of the various factors involved in the cleaning in order to ensure that their effect on the final percent recovery measurement is well understood. It is typically very cumbersome to deconvolute an aberrant percent recovery result ‘after-the-fact’ for a method that may have been in use over a long period of time. Cleaning Validation is a complex activity requiring a careful choice of sampling procedure and analytical method. It is therefore highly recommended to always use only the highest quality materials for swabs, filters, and solvents in cleaning validation protocols in order to assure that they cannot serve as sources of aberrant results, if and when those results do occur.
Both HPLC and TOC are highly sensitive methods that serve as assays for cleaning validation protocols. HPLC by its very nature is a specific assay in that it can identify peaks and assign them to specific residues, while TOC is a classically non-specific measure of overall carbon burden in a given environment.
Since these assays are both quantitative, typical analytical parameters such as accuracy, precision, linearity, detection, and quantitation limits must be evaluated as part of method development. While HPLC is a very commonly used tool in the pharmaceutical industry, the complexity, trace level sensitivity, and criticality of the cleaning validation protocol to drug safety merits special attention to the results from HPLC analysis. It is important to avoid using materials that might serve as sources of contamination through interference with the UV spectrum, or the detector of choice. In the event that such interference in the assay is unavoidable, understanding and perhaps even quantitating the interference so that the cleaning validation protocol is appropriately “science based” would pass muster under an investigation.
Attempts should be made to identify any additional peaks that appear in the chromatograms of swab extracted samples besides those arising from the expected residues. TOC (Total Organic Carbon) is a conductometric assay that correlates with carbon concentration, which provides an overall, non-specific estimate of residue burden left behind on the surface from a previous batch run. TOC measurements are highly sensitive and typically reported at the part per billion (ppb, or μg/L) level. As such, great care must be taken during the swab sampling and sample preparation to minimize external sources of organic carbon contamination.

SUMMARY

Cleaning validation is an essential step in the critical cleaning of pharmaceutical manufacturing environments. Swabbing is the preferred method of sampling such surfaces in the process of cleaning validation. The sampling and analysis methods have a direct and measurable impact on the percent recovery results from either HPLC or TOC assays. It is critical to ensure that the swab, filters, and associated materials used during the process are of the highest possible quality and do not contribute even trace levels of impurities that can interfere with the results.

References

1. Guide to Inspections of Validation of Cleaning Processes, FDA Office of Regulatory Affairs, Rockville, MD, 1993.
2. Carlson, J. “Is swabbing a regulatory requirement?” Journal of GXP Compliance, (14):1, 2010.
3. Pluta, P. and Sharnez, R., “Avoiding Pitfalls of Cleaning Validation.” Journal of GXP Compliance, (14):3, 2010.
4. McCormick, D. “Poor OOS Review Leads Causes of FDA Citations.” Pharma Technol. Oct 2005
5. ICH Guidance for Industry, Q7A; Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients, Aug 2001.
6. Yang, P, Burson, K., Feder, D. and Macdonald, F. “Method Development of Swab Sampling for Cleaning Validation.” Pharmaceutical Technology, Jan 2005.

Verification and Validation in Pharmaceutical Quality Assurance

If you’re studying pharmaceutical quality assurance, you’ve probably encountered the concept of validation and verification. Though both are essential for drug manufacturing or any good quality management system, it is vital not to confuse the two terms. One refers to being sure that the end product is what is expected with a particular process, while the other focuses on if the process is being done correctly. They are a key focus in a pharmaceutical quality control diploma and understanding them is crucial to your education.
Verification
Verification proceeds validation, but it does not replace it. In this part of the manufacturing or research process, equipment, products, production methods or programs must be repeatedly tested. This proves they are reliable. Though there will always be a margin for error included in any calculations, after doing the verification stage you can say you’ve completed this side of pharmaceutical quality assurance to the best of your ability.
That means you will look into not only how it was put together and assembled, but also the people operating it and the conditions it is operating under. In medical manufacturing, whether it is pills or prosthetics, nothing may be left up to chance. That might mean strict temperature and humidity regulation or meticulous logging of the body motions of staff when they do a task.
Validation
Once everything has been verified, you may use the data to validate it. Validation looks more at the end product, basically making sure that the same thing results every time the process you verified is followed. In drug testing or even food safety certificate, this will involve everything from weighing samples to mandatory mechanical tests of permeability and chemical composition.
In both cases, who does what may change the term that applies. For example, regardless of who does what, pharmaceutical quality assurance requires four processes in regards to the tools you use:

• Design Qualification (DQ)
• Installation Qualification (IQ)
• Operational Qualification (OQ)
• Performance Qualification (PQ)

While the latter three are usually subject to verification and validation by the manufacturer, DQ is generally handled by the vendor, a separate entity. In that case it is verification. If they don’t, then in the manufacturer’s hands, the process of quality control becomes validation. This may seem complicated, but keep in mind that for example, a pill cutting machine, is the product of a separate manufacturing process. Ideally verification and validation runs all the way to the raw materials that went into the equipment and product. This is not excessive, because trace contamination is impossible to control for. Imagine the metal bolts that hold a machine to the factory floor.  If one particular batch of steel has less tensile strength than another, small irregularities will enter the process. Maybe one machine will be less steady than another, and make more errors leading to lopsided drug dosages. It’s no wonder pharmaceutical quality control students devote so much time to the subject!

Validation of Analytical Assays and Test Methods for the Pharmaceutical Laboratory

By Robert V. Sarrio and Loui J. Silvestri, PhD
AccuReg
Overview
Analytical procedures used to measure the quality of pharmaceutical products span almost the entire range of currentlyavailable technologies and techniques. From immunoassay and electrophoretic techniques used to characterize protein moeities, and chromatographic and potentiometric methods used to evaluate the qualities of small molecules, the variety of procedures (and approaches necessary to prove these methods' validity and usefulness) can be overwhelming. However, when evaluating available procedures to determine which are best for your intended use, it is important to keep in mind that the most important aspect of any analytical method is the quality of the data it ultimately produces.
Perhaps the most useful and widely-consulted guidance in the industry is the USP's General Chapter 1225entitled, "Validation of Compendial Methods". This Chapter opens by referencing the Federal Food, Drug and Cosmetics Act (and hence, stressing the legal status of USP test procedures), then continues with a formal definition of "validation" as it applies to analytical methods. Directly quoted, the Chapter states that "Validation of an analytical method is the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications."
The most significant point raised by this definition is that the validity of a method can be demonstrated only through laboratory studies. It is not sufficient to simply review historical results; instead, laboratory studies must be conducted which are intended to validate the specific method, and those studies should be pre-planned and described in a suitable protocol. The protocol should clearly indicate the method's intended use and principles of operation, as well as the validation parameters to be studied, and a rationale for why this method and these parameters were chosen. The protocol also must include pre-defined acceptance criteria and a description of the analytical procedure, written with sufficient detail to enable persons "skilled in the art" to replicate the procedure.
Validation Parameters - Assays
USP General Chapter 1225, as well as the ICH Guideline for Industry (Text on Analytical Procedures), provide cursory descriptions of typical validation parameters, how they are determined, and which subset of each parameter is required to demonstrate validity, based on the method's intended use. For example, it would be inappropriate to determine limits of detection or quantitation for an active ingredient using an assay method intended for finished product release. However, if the method was intended to detect trace quantities of the active ingredient for purposes of a cleaning validation study, then knowledge of the detection and quantification limits are appropriate and necessary. For this reason, validation of each assay or test method should be performed on a case-by-case basis, to ensure that the parameters are appropriate for the method's intended use. This is even more important when validating stability-indicating assay methods, because these validations are more complex - for example, they may require forced degradation, samples spiked with known degradates, literature searches, etc.
The following definitions, taken from the ICH Guideline for Industry (Text on Analytical Procedures), will provide a background for subsequent discussion:
Analytical Procedure.
The analytical procedure refers to the way of performing the analysis. It should describe in detail the steps necessary to perform each analytical test. This may include, but is not limited to, the sample, the reference standard and the reagents preparations, use of the apparatus, generation of the calibration curve, use of the formulae for the calculation, etc.
Specificity.
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedure(s).
This definition has the following implications:

• IDENTIFICATION: To ensure the identity of an analyte.
• PURITY TESTS: To ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte, i.e., related substances test, heavy metals, residual solvents content, etc.
• ASSAY (Content or Potency): To provide an exact result which allows an accurate statement on the content or potency of the analyte in a sample.

Accuracy.
The closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value, and the value found.
Note: When measuring accuracy, it is important to spike placebo preparations with varying amounts of active ingredient(s). If a placebo cannot be obtained, then a sample should be spiked at varying levels. In both cases, acceptable recovery must be demonstrated.
Precision.
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.
Precision should be investigated using homogeneous, authentic (full scale) samples. However, if it is not possible to obtain a full-scale sample it may be investigated using a pilot-scale or bench-top scale sample or sample solution.
The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. Refer to this month's "The Regulatory Clinic" for a discussion of AccuReg's consensual interpretations of the following terms that express precision:
a. Repeatability. Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.
b. Intermediate Precision. Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.
c. Reproducibility. Reproducibility expresses the precision between laboratories (collaborative studies usually applied to standardization of methodology).
Detection Limit.
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.
Quantitation Limit.
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.
Linearity.
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.
Note: Measurements using clean standard preparations should be performed to demonstrate detector linearity, while method linearity should be determined concurrently during the accuracy study. Classical linearity acceptance criteria are 1) that the correlation coefficient of the linear regression line is not more than some number close to 1, and 2) that the y-intercept should not differ significantly from zero.
When linear regression analyses are performed, it is important not to force the origin as (0,0) in the calculation. This practice may significantly skew the actual best-fit slope through the physical range of use.
Range.
The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Robustness.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters and provides an indication of its reliability during normal usage.
Note: Ideally, robustness should be explored during the development of the assay method. By far the most efficient way to do this is though the use of a designed experiment. Such experimental designs might include a Plackett-Burman matrix approach to investigate first order effects, or a 2k factorial design that will provide information regarding the first (main) and higher order (interaction) effects.
In carrying out such a design, one must first identify variables in the method that may be expected to influence the result. For instance, consider an HPLC assay which uses an ion-pairing reagent. One might investigate: sample sonication or mixing time; mobile phase organic solvent constituency; mobile phase pH; column temperature; injection volume; flow rate; modifier concentration; concentration of ion-pairing reagent; etc. It is through this sort of a development study that variables with the greatest effects on results may be determined in a minimal number of experiments.
The actual method validation will ensure that the final, chosen ranges are robust.

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Other points to consider include:
System Suitability
In addition, prior to the start of laboratory studies to demonstrate method validity, some type of system suitability must be done to demonstrate that the analytical system is performing properly. Examples include: replicate injections of a standard preparation for HPLC and GC methods; standardization of a volumetric solution followed by assays using the same buret for titrimetric methods; replicate scanning of the same standard preparation during UV-VIS assays, etc. When the method in question utilizes an automated system such as a chromatograph or an atomic absorption spectrophotometer, a suitable standard preparation should be intermittently measured during the sample analysis run. The responses generated by the standard should exhibit a reasonable relative standard deviation. This is done primarily to demonstrate the stability of the system during sample measurements. System suitability for dissolution studies should be performed using both USP non-disintegrating and disintegrating tablets prior to the validation of dissolution methods.
Validity Checks - General Tests
It is important to realize that assays are not the only tests important in evaluating the qualities of a drug product. The USP contains numerous identity tests of a chemical nature. In these types of tests, one should treat a placebo preparation with the reaction reagent to ensure a negative result is achieved. Otherwise, the test has no meaning. Dissolution tests, for instance, should be evaluated for adequate sink conditions (i.e., adequate solubility in an adequate volume of the dissolution media) prior to development.
Protocols
As mentioned earlier, prior to initiating a validation study, a well-planned validation protocol should be written and reviewed for scientific soundness and completeness by qualified individuals. The protocol should describe the procedure in detail, and should include pre-defined acceptance criteria and pre-defined statistical methods. Following approved by the appropriate corporate and Quality Control authorities, the protocol should be executed in a timely manner. A typical assay validation will require the preparation of product placebo(s), standards, and many samples.
How many times should an assay be repeated to ensure "validity"? Although 3 sequential replicates are often considered the "magic number," a far more definitive number is one produced by a sound scientific rationale, usually with the assistance of statistical analyses.
Subsequent to the execution of the protocol, the data must be analyzed with results, conclusions and deviations presented in an official validation summary report. Provided the pre-defined acceptance criteria are met, and the deviations (if any) do not affect the scientific interpretation of the data, the method can be considered valid. A statement of the method's validity should be placed at the beginning of the final summary report, along with the signatures and titles of all significant participants and reviewers.

Process Validation : An Overview

• "Validation is a systematic approach to gathering and analyzing sufficient data which will give reasonable assurance (documented evidence), based upon scientific judgment, that a process, when operating within specified parameters, will consistently produce results within predetermined specifications."

• “Process validation is establishing documented evidence which provides a high degree of assurance that a specific process (such as the manufacture of pharmaceutical dosage forms) will consistently produce a product meeting its predetermined specifications and quality characteristics.”

• “Process validation is defined as the collection and evaluation of data, from the process design stage through commercial production, which establishes scientific evidence that a process is capable of consistently delivering quality product.”

• “Validation is the act of demonstrating and documenting that a procedure operates effectively. Process validation is the means of ensuring and providing documentary evidence that processes (within their specified design parameters) are capable of consistently producing a finished product of the required quality.”

Importance of Validation

• Increased throughput
• Reduction in rejections and reworking
• Reduction in utility costs
• Avoidance of capital expenditures
• Fewer complaints about process-related failures
• Reduced testing in-process and in finished goods
• More rapid and reliable start-up of new equipment
• Easier scale-up from development work
• Easier maintenance of equipment
• Improved employee awareness of processes
• More rapid automation

Stages/Phases of Validation

Stage 1: Process Design (Pre-Validation Phase)

This is the step where building and capturing of the process knowledge and understanding took place. Early design of processes and experiments should be performed during this stage. It covers all activities relating to product research and development, formulation, pilot batch studies, scale-up studies, transfer of technology to commercial scale batches, establishing stability conditions, storage and handling of in-process and finished dosage forms, equipment qualification, installation qualification, master production documents, operational qualification, process capability. Also this is the stage in which the establishment of a strategy for process control is taking place using accumulation knowledge and understanding of the process.

Stage 2: Process Qualification (Process Validation Phase)

This stage is confirmation that the process design is capable of reproducing the manufacturing process. It confirms that all established limits of the Critical Process Parameters are valid and that satisfactory products can be produced even under “worst case” conditions. GMP compliant procedures must be followed in this stage and successful completion of this stage is necessary before commercial distribution of a product.

Stage 3: Continued Process Verification (Validation Maintenance Phase)

The Validation Maintenance Stage requires frequent review of all process related documents, including validation audit reports to assure that there have been no changes, deviations, failures, modifications to the production process, and that all SOPs have been followed, including change control procedures. Before any batch is distributed for marketing, the manufacturer must have full assurance of its performance. A successful validation program depends on the knowledge and understanding and the approach to control manufacturing processes. These include the source of variation, the limitation of the detection of the variation, and the attributes susceptible of the variation. It is the responsibility of the manufacturer to judge and provide evidence of a high degree of assurance in its manufacturing processes. They are also responsible for maintaining the degree of assurance accomplished, even if some minor changes occurred due to personnel, material and process changes.

Type of Validation

• Prospective Validation 

is process of gathering of data and documentary evidence about a product and its process before it is sent to market or for distribution, a new product validation or a validation after making a change in the master formula, to determine if the product meet its predetermined standards. The process starts with designing of batch record , raw material specification and in process specifications and limits ,establishing sampling plan, and equipment lists and required environment controls.

• Concurrent Validation 

Is a validation process where in current production batches are used to confirm the compliance of processing parameters and standards. Concurrent validation is the set of validation procedures following prospective validation. Concurrent validation is carried out for ensuring the product batches produced in prospective validation meet the required standards and quality so that those can be distributed in market. 
Concurrent validation is of great use when the test employed is not destructive and can determine the product meet predetermined standards and quality. Concurrent validation is also carried out on product or process which is previously validated process to ascertain that the product or the process meets required standards and is validated.

• Retrospective Validation 

 is a type of validation where in the product is already and established process such batches of products which are being sent to market are studied to gather documentary evidence about the efficacy of the process or any in process tests and the product it self, such type of validation can be adapted to validate product and the process even if the product and process is not validated earlier. It also makes use of data from old batches to establish the efficacy and compliance of product or process standards. 

•  Re validation

To ensure that changes in the process and/or in the process environment, whether intentional or unintentional, do not adversely affect process characteristics and product quality.

Re-validation may be divided into two broad categories:

• Re-validation after any change having a bearing on product quality.
• Periodic re-validation carried out at scheduled intervals.