Analytical techniques for biopharmaceutical development

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When all analytical tests are completed, the manufacturer creates the CoA by extracting the relevant information from the database. Only a subset of the results, which are defined by specifications, will be listed on the CoA. The specifications will depend on the extent of peak characterization and the clinical significance of the various peaks Apostol, Schofield et al.

Therefore, the list will change evolve with the stage of drug development. In such a context, LOQ should be considered as the analyte specific value expressed in units of protein concentration, a calculation for which instrument sensitivity cannot be disregarded in contrast to LOD estimation.

The signal created by the analyte may vary with the load, while the relative percentage of the analyte does not change. This creates a situation where the analyte of interest can be hidden within the noise or, alternatively, can be significantly above the noise for the same sample analyzed at two different load levels within the range allowed by the method.

The above equation expresses LOQ as a function of signal-to-noise ratio and the observed purity of the analyte. Both parameters can change from test-to-test, due to equipment variability and sample purity variability. Therefore this equation should be viewed as the dynamic live assessment of LOQ.

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System suitability is intended to demonstrate that all constituents of the analytical system, including hardware, software, consumables, controls, and samples, are functioning as required to assure the integrity of the test results. However, guidance is vague and reference is often made to Pharmacopeias for additional information. The USP, EP and JP contain guidance for a broad scope of HPLC assays, including assays of the active substance or related substances assays, assays quantified by standards external or internal or by normalization procedures, and quantitative or limit tests.

While each type of assay is described in the compendia, the specific system suitability parameters to be applied for each type of assay, is not included with the description. Thus, some interpretation is required. The interpretation of how to best meet the requirements of the various compendia while still maintaining operational efficiency is a significant challenge for industry. Existing guidance for system suitability was developed for pharmaceutical compounds and may not be directly applicable for proteins which, due to their structural complexity and inherent heterogeneity, require additional considerations beyond those typically required for small molecules.

For example, appraisal of resolution by measuring the number of theoretical plates commonly done for small molecules , may not be the best way to assess the system readiness to resolve charge isoforms of a protein on an ion exchange column. This may be due to the relatively poor resolution of protein peaks resulting from inherent product microheterogeneity, when compared to the resolution typically seen with small molecules. However, this methodology the number of theoretical plates may be a very good indicator to measure the system performance for size exclusion chromatography SEC , which does not typically resolve product isoforms resulting from microheterogeneity.

To appropriately establish system suitability, we need to consider both the parameter that will be assessed and the numerical or logical value s , generally articulated as acceptance criteria, associated with each parameter. System suitability should be demonstrated throughout an assay by the analysis of appropriate controls at appropriate intervals.

It is a good practice to establish the system suitability parameters during method development, and to demonstrate during qualification that these parameters adequately evaluate the operational readiness of the system with regard to such factors as resolution, reproducibility, calibration and overall assay performance. Prior to validation, the system suitability parameters and acceptance criteria should be reviewed in order to verify that the previously selected parameters are still meaningful, and to establish limits of those parameters, such that meaningful system suitability for validation is firmly established.

One important issue that merits consideration is that the setting of appropriate system suitability parameters is a major contribution to operational performance in a Quality environment, as measured by metrics such as invalid assay rates. A key concept is that the purpose of system suitability is to ensure appropriate system performance including standards and controls , not to try to differentiate individual sample results from historical trends e.

In practice, setting system suitability parameters that are inappropriately stringent can result in the rejection of assay results with acceptable precision and accuracy. ICH Q2R1 prescribes that the evaluation of robustness should be considered during the development phase. The robustness studies should demonstrate that the output of an analytical procedure is unaffected by small but deliberate variations in method parameters.

Robustness studies are key elements of the analytical method progression and are connected to the corresponding qualification studies. Method robustness experiments cannot start before the final conditions of the method are established. It is a good practice to identify operational parameters for the method and to divide them in the order of importance into subcategories according to their relative importance, which are exemplified below:.

It is highly impractical to evaluate the impact of all possible parameters on the output of the method. It is a good practice to prospectively establish a general design outline for such studies. The studies may be carried out using the one-factor-at-a-time approach or a Design of Experiment DOE approach. The selection of assay parameters can vary according to the method type and capabilities of the factorial design, if applicable.

The maximum allowable change in the output of the analytical method can be linked to the target expectations for the precision of the method, which are derived from the Horwitz equation Horwitz ; Horwitz and Albert ; Horwitz and Albert Recently a number of software packages have become available to assist with the design and data analysis Turpin, Lukulay et al. Remediation of validated analytical methods is typically triggered by the need to improve existing methods used for disposition of commercial products.

The improvement may be required due to an unacceptable rate of method failures in the GMP environment, lengthy run times, obsolete instruments or consumables, the changing regulatory environment for specifications or stability testing, or for other business reasons. We anticipate that technological advances will continue to drive analytical methods toward increasing throughput. In this context, it appears that many release methods are destined for change as soon as the product has been approved for commercial use Apostol and Kelner ; Apostol and Kelner This is due to the fact that it takes more than 10 years to commercialize a biotechnology drug, resulting in significant aging of the methods developed at the conception of the project.

Therefore, the industry and regulators will need to continuously adjust strategies to address the issue of old vs. Frequently, old methods have to be replaced by methods using newer technologies, creating a significant challenge for the industry in providing demonstration of method equivalency and a corresponding level of validation for the methods. When we consider the critical role that analytical method development, qualification and validation play in the biopharmaceutical industry, the importance of a well designed strategy for the myriad analytical activities involved in the development and commercial production of biotechnology products becomes evident.

The method qualification activities provide a strong scientific foundation during which the performance characteristics of the method can be assessed relative to pre-established target expectations. This strong scientific foundation is key to long-term high performance in a Quality environment, following the method validation, which serves as a critical pivotal point in the product development lifecycle.

As noted previously, the method validation often serves as the point at which the Quality organization assumes full ownership of analytical activities. If done properly, these activities contribute to operational excellence, as evidenced by low method failure rates, a key expectation that must be met to guarantee organizational success.

Without the strong scientific foundation provided by successful method development and qualification, it is unlikely that operational excellence in the Quality environment can be achieved. As analytical technologies continue to evolve, both the biotechnology industry and the regulatory authorities will need to continuously develop concepts and strategies to address how new technologies impact the way in which the Quality by Design principles inherent in the analytical lifecycle approach are applied to the development of biopharmaceutical products.

The ICH Q2 guideline requires that an analytical method be validated for commercial pharmaceutical and bio-pharmaceutical applications. This concern is exacerbated by the requirement for modern pharmaceutical and biopharmaceutical companies to seek regulatory approval in multiple jurisdictions, where the instrumentation, consumables, and scientific staff experience at the testing location may be very different than that present in the place where the drug was developed. These considerations raise questions about the value of the current format of the validation studies conducted by the industry.

Moreover, it is not clear how the validation data obtained using existing methodologies should or even could be used toward the assessment of the uncertainty of the future results, given the many factors that contribute to the uncertainty. Perhaps the time is right for the industry to consider the use of a combination of sound science and reasonable risk assessment to change the current practice of the retrospective use of method validation to the new paradigm of live validation of purity methods based on the current information embedded in the chromatogram.

Laboratories that work in a GMP environment are required to produce extensive documentation to show that the methods are suitable. Pharmaceutical and biopharmaceutical companies thoroughly adhere to these requirements, inundating industry with an avalanche of validation work that has questionable value toward the future assessment of uncertainty. The predication of uncertainty provides an alternative that has the potential to reduce the work required to demonstrate method suitability and, in turn, provide greater assurance of the validity of the results from the specific analysis in real time.

The establishment of qualification target expectations can be considered as a form of Quality by Design QbD , since this methodology establishes quality expectations for the method in advance of the completion of method development. Also, the analytical lifecycle described here covers all aspects of method progression, starting with method development, the establishment of system suitability parameters, and qualification and robustness activities, culminating in method validation, which confirms that the method is of suitable quality for testing in Quality laboratories.

The entire analytical lifecycle framework can be considered as a QbD process, consistent with evolving regulatory expectations for pharmaceutical and biopharmaceutical process and product development. Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers. Login to your personal dashboard for more detailed statistics on your publications.

Edited by Ira S. Edited by Jeevan Prasain. We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists.


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Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. Downloaded: Introduction Method validation has a long and productive history in the pharmaceutical and now, biopharmaceutical industries, but it is an evolving discipline which changes with the times. They did not ask to see some evidence of repeatability, intermediate precision and other performance characteristics, a situation that would not be permissible in the industrial world due to regulatory requirements for method validation Method validation in the pharmaceutical and biopharmaceutical industries is designed to help ensure patient safety during clinical trials and later when the drug becomes commercialized.

Method validation for the biotechnology industry The development of biotherapeutics is a complex, resource-intensive and time-consuming process, with approximately 10 years of effort from target validation to commercialization. Qualification and validation of release and stability methods Qualification should be performed prior to method implementation in the Quality laboratories to ensure the integrity of the data provided on the Certificate of Analysis for clinical lots.

In the context of the analytical lifecycle, the key components of method validation are as follows: The experimental design of method validation should mimic the qualification design, and acceptance criteria should be linked to the target expectations used in the qualification experiments. This typically includes the following sample types: Sample types listed on all release and stability specifications intermediates, drug substance and drug product ; Samples associated with process controls and in-process decision points.

Performance characteristics In order to produce a reliable assessment of method performance, all necessary performance characteristics should be evaluated in carefully designed experiments. Table 1. Table 2. Accuracy The determination of accuracy for protein purity methods presents significant challenges. Linearity and range Linearity and range are typically assessed in a complex experiment demonstrating a linear change of peak area with analyte concentration.

Specificity The specificity of analytical methods is typically assessed by examining system interference with the detection and quantification of analytes. System suitability System suitability is intended to demonstrate that all constituents of the analytical system, including hardware, software, consumables, controls, and samples, are functioning as required to assure the integrity of the test results. Method robustness ICH Q2R1 prescribes that the evaluation of robustness should be considered during the development phase.

Challenges associated with validated methods Remediation of validated analytical methods is typically triggered by the need to improve existing methods used for disposition of commercial products. Concluding remarks When we consider the critical role that analytical method development, qualification and validation play in the biopharmaceutical industry, the importance of a well designed strategy for the myriad analytical activities involved in the development and commercial production of biotechnology products becomes evident.

More Print chapter. How to cite and reference Link to this chapter Copy to clipboard. Available from:. Already less labour-intensive than many alternative spectroscopy based technologies, DSC is now increasingly automated, both in terms of measurement capacity and data analysis.

Sample volume and working concentration are challenges that have also been addressed, with modern systems offering minimal sample consumption and operating at therapeutic concentrations of the biologic, bringing closer the goal of at-line QC testing of CQAs. Automation has multiple benefits, not least in increasing throughput and reducing operator time and expertise, but also in allowing improved and non-subjective analysis. Automated and quantitative analysis tools allow the extraction of maximum information from the data produced.

It helps to generate results that are easily understood and readily reconciled with data from complementary orthogonal techniques. Coupled with all of this is the facility for robust method development and transfer, contributing to increased productivity and greater consistency and applicability throughout the biopharmaceutical development workflow, with method transfer now possible across and between labs and sites.

As analytical instruments become more closely aligned with the needs of the biopharmaceutical industry, these same requirements will be met by data analysis and management, with new functionalities enabling the rationalisation of large datasets that have been generated using orthogonal and complementary techniques. This will be a critical step towards the full implementation of the QbD approach. Microcalorimeter resets standard for characterising protein and biomolecule stability.

Validation support for laser diffraction particle sizing. New automated tool for biophysical characterisation. Developing analytical instrumentation for the biopharmaceutical industry Nov Equipment Research Ensuring that analytical instrumentation readily meets the continually evolving requirements of biopharmaceutical research, development and manufacturing is an ongoing challenge. Companies Malvern Instruments Ltd. Related Articles Malvern Instruments establishes new biopharmaceutical applications laboratory in San Diego.

US researchers work on novel polymers for drug delivery. Introducing morphologically directed Raman spectroscopy. Faster de-formulation of dry powder inhalers. Process development of biopharmaceuticals is particularly challenging because biomolecules are too complex to be manufactured by traditional chemical synthesis. Biopharmaceuticals produced by living cells or cultures can be heterogeneous and exhibit characteristics that can change over time even if the same system is used to generate the product.

For small molecules, analytical techniques can be used at the end of the production process to characterize dene the product. Because of the complexity of biopharmaceuticals, this approach is difcult to implement. Instead, it is hypothesized that a consistent manufacturing process will yield a consistent product. So, for the production of biologics, more emphasis is placed on the manufacturing details which encompass the chemistry, manufacturing, and controls section of regulatory applications.

The more robust a manufacturing process, the less need for characterization of the end product. Other concepts of high importance in biopharmaceutical development are formulation, stability, and delivery. This is because proteins are highly complex biomolecules that are sensitive to their environment dened by the drugs formulation and storage. A formulation is developed in the preclinical stage and evaluated continuously until nal approval of the product. The key aspects of formulation are based on determination of the stability of the drug in the presence of particular conditions or excipients or both.

Usually accelerated stability and intended storage conditions studies are performed. In these assays, the effect of exposure to physical and chemical agents such as heat and light on the drug is evaluated. These studies require techniques capable of resolving impurities generated during exposure of the sample to harsh conditions.

Such methods are said to be stability-indicating. These methods may be the same or different from those used to resolve and detect impurities generated during production of the drug. It is important for development scientists to familiarize themselves with the regulatory process, which denes the development stages of a biopharmaceutical. Along this path there are several checkpoints that must be passed before reaching the next plateau. These checkpoints or phases affect all groups within. For example, for methods development and characterization scientists, the required level of knowledge on the behavior of methods in establishing the structure, purity, potency, and stability of the drug increases as the process advances.

Because bioanalytical methods constantly improve, development scientists ability to nd impurities increases. The cycle could go on forever, and a drug may never be considered truly pure. Developers must strike a balance between creating a process that is far too complex and expensive both for the manufacturer and ultimately for the patients and one that will produce a safe drug. The concepts are presented here to position everyday work within the greater perspective of drug development. Drug discovery Although some drugs are developed by fairly large biotechnology companies, most of the promising drug candidates in development were discovered in academic research laboratories, often as a result of disease investigation, and not because of active research pursuing particular drugs for their activity.

It is common to identify the individuals who discover the drug as part of the founders of a company. At this stage, drugs are usually produced in small quantities used for activity studies. Some characterization and analytical drug testing is necessary to ensure that the observed results are due to the drug itself and that the observed activity is reproducible. Preclinical development Preclinical development is charged with dening the initial safety and activity proles of promising new drugs. The industry is hard at work developing alternative systems to evaluate drugs, but at present the bioactivity and efcacy of a protein therapeutic can only be determined through testing in biological systems animal studies.

One of the rst characteristics to be evaluated after activity is the toxicity prole and pharmacokinetics of the drug. Toxicity studies are used to determine the safe range of dosing for initial phase I clinical trials in humans. Pharmacokinetics studies provide data on absorption, distribution, metabolism, and excretion ADME of the drug. At least two different species of animals typically, the early studies are performed in rodents, and the late, more expensive studies in nonhuman primates are used in toxicity testing of biopharmaceuticals.

At the preclinical stage, the production group actively evaluates processes that are potentially suitable for the generation of the lead molecule. Communication between the preclinical and process development groups is crucial because production modications may result in activity changes. The portfolio of techniques, which is a work in progress at this stage, is used to continue product characterization and often to evaluate discrepancies in activity e.

Investigational new drug application There are two major regulatory documents in the life of a pharmaceutical biological product. The IND document is required because companies cannot administer drugs to humans without FDA authorization; thus, the IND application is a companys request to regulatory bodies to allow the exposure of volunteers and patients to the drug under study.

The IND designation is a living document in the sense that there is ow of information during its different stages of development. It is in effect until the approval of the drug for commercialization at which point a BLA is led or until the company decides to stop clinical trials for the drug. The drug development phases are aimed at determining the safety prole, dosage range, clinical end points, ADME, and effectiveness efcacy of the drug candidate.

Drug development is a Process, and therefore information, data, and knowledge are accumulated over time. Thus, it should be anticipated that many things will be reevaluated as the drug progresses from one phase to another and that unforeseen issues may result that require resolution before continuation of the studies. Phase I clinical development Phase I clinical development is carried out in a relatively small group of volunteers and patients usually 15 to , where the main goal of the trial is to establish the safety characteristics of the molecule.

Because the number of volunteers is low, only frequent adverse effects are observed. It is also common to explore dose range and dose scheduling during phase I. Often doses below the expected treatment level are used rst for safety reasons and the amount of drug is increased over time. This phase is initiated 30 days after submission of the IND to the FDA, unless the regulatory agency has concerns about toxicity or the design of the study.

If this occurs, the clinical trial is put on hold until the issue is resolved. Clinical trials are usually double-blinded the clinicians and patients do not know if the substance administered contains drug or placebo. The trials are blinded to minimize the so called placebo effect, in which patients respond to the treatment even in the absence of true drug effect. This response can be positive the patient feels better or negative the patient has adverse effects , and thus can blur the true benets and risks of the biopharmaceutical.

During phase I the analytical laboratory continues characterization of the drug molecule and optimization and renement of the methodology. Production is also rening the process to increase purity and yield and make it amenable to scaling up. Formulation studies usually consist of excipient screening during this phase.

Analytical techniques for biopharmaceutical development /

Phase II clinical development Phase II involves a greater number of patients usually to than phase I clinical studies. At this stage more emphasis is placed on activity, dosing, and efcacy than in phase I, and thus, only patients are used for phase II studies and beyond. Sometimes, reevaluation. Just as in phase I, phase II studies are usually blinded, but at this stage a control group and multiple centers of study may be added depending on the complexity of the trial, which in turn depends on the observations achieved during phase I.

Phase II studies are also useful in identifying populations that will be more likely to benet from the treatment. Because the number of subjects is higher, phase II studies can reveal adverse effects that are less common but not large enough to gather unambiguous statistical information to prove efcacy and safety. The production and purication groups continue to evaluate raw materials and purication processes and to perform lot-release tests.

Analytical Techniques for Biopharmaceutical Development

More emphasis is placed on manufacturing scale-up as phase II studies progress. Quality assurance, quality control, compliance and regulatory affairs, and clinical development are actively preparing to organize the package of information describing drug characteristics and activity data, in preparation for the post-phase II meeting with the regulatory agencies. The trial can be designed to provide data that support the licensure to market the drug pivotal trial , or it can be used to further dene the characteristics of the molecule in a clinical setting e.

The number of patient volunteers needed for phase III trials depends on many factors, but most studies enroll to individuals. The goal of phase III studies is to gather enough evidence on the riskbenet relationship in the target population. In this phase, long-term effects are analyzed for drugs that are intended for multiple- or extended-time usage. Phase III trials are expensive, and therefore only drugs with a very high potential for commercialization are evaluated.

During phase III the analytical laboratory performs systematic methods validation and continues with product characterization. A suitable formulation or a formulation candidate is in place and testing for stability continues. Production evaluates the consistency of the manufacturing process, which should be at a scale capable of delivering commercial quantities.

Advanced studies are continued or initiated to evaluate chronic toxicology and reproductive side effects in animal models. Biologics license application BLA In this document, nonclinical, clinical, chemical, biological, manufacturing, and related information is included. The goal of the manufacturer is to demonstrate that the drug is safe and effective, and the manufacturing and quality control are appropriate to ensure identity, strength, potency and purity, consistency of the process, and adequate labeling.

The BLA is supported by all the data collected during the clinical trials, but. The BLA is a request to market a new biologic product, and it contains data which demonstrate that the benets of the drug outweigh any adverse effects. Because a large amount of information needs to be reviewed and therefore presented in a clear manner , a pre-BLA meeting is scheduled with regulatory agencies. Phase IV clinical surveillance When the drug has reached the market, further studies are conducted to create proles on adverse effects, evaluate the drugs long-term effects, and further tune dosage for maximum efcacy.

- Analytical Techniques for Biopharmaceutical Development by Rodriguez D.

Potential interactions with other therapies are monitored closely. By observing the behavior of the drug after introduction to the general public or by extending the use of the product to populations not included in the trials, sometimes additional indications are uncovered or conrmed. Safety and efcacy comparisons to existing therapies may also be performed. Anne Montgomery, editor-in-chief, Haystead, editor-in-chief, Advanstar. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably conrm the most frequently performed assay to be protein concentration.

In the s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In his method was published in the Journal of Biological Chemistry.


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In the Institute for Scientic Information ISI reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist.

As described in the following chapter, there are many biopharmaceutical applications of protein assays. Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the rst task for subsequent analytical procedures because assays for purity, potency, or identity require that the protein concentration be known.

Hence it is typical for several different methods to be employed under the umbrella of protein concentration measurement, depending on the requirements of speed, selectivity, or throughput. The protein concentration is valuable as a stand-alone measurement for QC and stability of a protein. However, protein concentration methods provide no valuable Fortunately, protein concentration methods are relatively simple low-tech and inexpensive. The simplest assays require only a spectrophotometer calibrated for wavelength and absorbance accuracy, basic laboratory supplies, and good pipetting techniques.

Protein concentration assays are quite sensitive, especially given the typical detection limits required for most biopharmaceuticals. What follows is not an exhaustive or up-to-the-minute survey of the methods available for protein quantitation, but a practical guide to selecting the appropriate assay for each stage of drug development. A case study further illustrates the application of the standard protein methods to the drug development process.

The reader is referred to reviews on the topic for further details. A brief summary of the principles, advantages, and limitations of these methods follows. Colorimetric Assays The colorimetric methods depend on a chemical reaction or interaction between the protein and the colorimetric reagent. The resulting generation of a chromophore, whose intensity is protein-concentration dependent, can be quantied using a spectrophotometer. Beers Law is employed to derive the protein concentration from a standard curve of absorbances.

Direct interaction of the protein with a chromogenic molecule dye or protein-mediated oxidation of the reporter molecule generates a new chromophore that can be readily measured in the presence of excess reagent dye. A common drawback of the larger sample volume is a greater potential for interference by the increased amounts of excipients present in the nal reaction volume.

Alternatively, techniques can be employed to increase the concentration of samples prior to analysis, usually with the added advantage that interfering excipients are removed in the process. One such example is protein precipitation with acetone or trichloroacetic acid. However, the additional sample handling will probably decrease the accuracy and precision of the nal result, and protein recovery studies should be performed. With respect to accuracy, these colorimetric methods require calibration of the absorbance of the chromophore that is created by the proteinreagent interaction.

This is typically achieved by preparing a standard curve with either a readily available standard protein or the target protein itself. If a standard protein such as bovine serum albumin BSA is to be used, a correction factor will need to be determined to generate an accurate value for the target protein. This can be achieved by using amino acid analysis to establish a true value for the target protein, comparing it with the value obtained for the target protein from the BSA standard curve, and then generating the correction factor for the BSA-derived value.

Clearly, sufcient replicates of both assays are necessary to generate an accurate correction factor. Once this has been done for a given colorimetric technique, target protein, standard protein, and a given set of assay conditions, an accurate target protein concentration can be obtained. It will be necessary to empirically calibrate the response of each new target protein to these reagents. Changes in the buffers and excipients will also require recalibration of the assay because the method may be sensitive to the buffer components. Lowry Method The Lowry method is probably the most widely used method for protein concentration.

The chemistry behind the method involves redox reactions. The target protein is treated with alkaline cupric sulfate in the presence of tartrate, which results in the reduction of the cupric ion by the protein and complexation of the resulting cuprous ion by the tartrate. This tetradentate cuprous ion complex is then reacted with FolinCiocalteau phenol reagent.

Reduction of this reagent by the cuprous complex yields a water-soluble blue product that has an absorbance at nm. This method only requires approximately 1 h of total incubation, but has the disadvantage of two incubations with exact incubation times. The practical limit for the number of samples per assay is approximately Interfering substances include detergents and reductants thiols, disuldes, copper chelators, carbohydrates, tris, tricine, and potassium ions.

The Lowry assay has a working range of 1 to g. The BioRad DC protein assay requires only a single min incubation, and absorbance is stable for at least 2 h. The microtiter plate assay procedures available for both protein concentration ranges provide automation for high throughput. The target protein is treated with alkaline cupric sulfate in the presence of tartrate, which results in the reduction of the cupric ion to cuprous by the protein.

The cuprous ion is then treated with bicichoninic acid BCA and two. BCA molecules complex with the cuprous ion to yield a water-soluble purple product that has an absorbance at nm. This method only requires 30 min of incubation at 37C but has the disadvantage of not being a true end-point assay because the color will keep developing with time.

In reality, the rate of color development is slowed sufciently following incubation to permit large numbers of samples to be assayed in a single run. The structure of the protein, the number of peptide bonds, and the presence of cysteine, cystine, tryptophan, and tyrosine have all been reported to be responsible for color formation. Interfering substances include reductants and copper chelators in addition to reducing sugars, ascorbic and uric acids, tyrosine, tryptophan, cysteine, imidazole, tris, and glycine.

Increasing the amount of copper in the working reagent can eliminate interfering copper-chelating agents. If the target protein is in a dilute aqueous formulation, the concentration can be determined with the micro BCA assay. These modications permit the detection of BSA at 0. The major disadvantage of these modications is that the presence of interfering substances decreases the signalto-noise ratio and thus the sensitivity of the assay. Bradford Method The Bradford method is probably the simplest colorimetric method, relying on only the immediate binding of the target protein to Coomassie Brilliant Blue G in acidic solution.

The water-soluble blue product has an absorbance at nm. Mechanistic studies suggest that the sulfonic acid form of the dye is the species that binds with the protein. Van der Waals and hydrophobic interactions are also believed to have a role in the binding. This method consists only of mixing with no requirement for incubation time or elevated temperatures. Detergents are the major interfering substances for this assay. Despite these advantages, the Bradford assay exhibits high interassay variability, which limits its use in situations where high precision is required.

Direct Absorbance Methods The direct absorbance methods require only a protein-specic extinction coefcient to deliver an accurate protein concentration. These methods typically require minutes to perform and require only a spectrophotometer and a good quantitative. In addition, these methods are amenable to automation.

They do not require a standard curve for quantitation but are protein composition and structure dependent. Absorbance methods typically rely upon the intrinsic absorbance of a polypeptide or protein at nm. The aromatic amino acids that absorb at this wavelength are tyrosine, tryptophan, and phenylalanine. Because these residues remain constant for a given protein, the absolute absorption remains constant. An extinction coefcient needs only to be determined once and is then absolute for the target protein in that buffer system. Determination of the extinction coefcient is a relatively straightforward task.

The target protein is diluted to give ve different concentrations. These samples are then divided into two aliquots. Amino acid analysis AAA accurately determines the protein concentration of one set of samples at the ve concentrations, and the absorbance at nm A is measured for the other set of samples. The slope of a plot of A vs. Fluorescence Methods Molecules with intrinsic uorescence absorb energy at a specic excitation wavelength ex and rise to an excited state.

The energy is released at a longer emission wavelength em as the molecules return to ground state. Fluorescence at distinct wavelengths where there is little interference from other sample components provides high selectivity for the uorescent molecules. In addition, sensitivity with these methods is high because there is little interference from background light at the emission wavelengths.

Native Fluorescence Native uorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at nm and emits at to nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a proteins native uorescence is feasible.

Another consideration is the proteins conformation, which directly affects its uorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native uorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native uorescence results in measurements specic for the protein in a buffer system or pH.

Consequently, protein denaturation may be used to generate more reproducible uorescence measurements. Derivatization with Fluorescent Probes Proteins that do not contain tryptophan or tyrosine must be derivatized prior to uorescence detection. A common derivatization chemistry involves the reaction.

The selectivity provided by the derivatization of the amines can be further enhanced by separation of the uorescent probes and derivatized sample components using an analytical method such as high-performance liquid chromatography HPLC. Alternatively, postcolumn derivatization can occur following separation of the target protein from other sample components. Fluorescent probes that react with other functional groups offer different selectivities.

Although derivatization with a uorescent probe may provide selectivity and sensitivity within a complex sample matrix, this labor-intensive method is less precise than direct measurement methods or even colorimetric assays that require less extensive sample preparation.

Tris interferes with amine derivatization, and care should be taken to determine if other buffer components affect the derivatization chemistry of choice. Amino Acid Analysis The fourth category of protein assay is amino acid analysis. This method is the most accurate and robust method for determination of protein concentration, but is appropriate only for pure proteins. In addition, it is relatively slow and requires specialized instrumentation and knowledge of the target proteins theoretical amino acid composition.

AAA usually involves hydrolysis of the protein into its constituent amino acids, which are then derivatized with a UV or uorescent label and quantied by HPLC against known amino acid standards. Hydrolysis occurs with strong acid at high temperatures. Hence some amino acids are modied e.

Peptide bonds between hydrophobic residues such as leucine, phenylalanine, or valine are hard to break and may require extended hydrolysis detrimental to the recovery of other amino acids. Although special hydrolysis conditions exist for the recovery of labile residues such as threonine, serine, tyrosine, and tryptophan, no one set of hydrolysis conditions quantitatively yields all amino acids.

After hydrolysis, the liberated amino acids are typically derivatized with phenylisothiocyanate PITC. Alanine, leucine, valine, and phenylalanine are among the most stable residues and are typically used for protein quantitation. There are few interfering substances because the method involves hydrolysis, derivatization, and chromatography with detection at a unique wavelength. Most excipients will not affect the hydrolysis step, but one has to be careful to ensure that the amino acids used to quantitate the protein are not destroyed.

In addition, it must be determined if the excipients interfere with the derivatization chemistry or the chromatography. A BSA standard in the same buffer formulation is routinely run in parallel to the target protein to ensure the accuracy of the method. Custom Quantitation Methods Finally, there are custom two-step quantitation methods such as chromatography or ELISA that require a capture step for isolating the protein and then a quantitation step based on a standard curve of the puried target protein.

The preliminary capture step may also concentrate the protein for increased sensitivity. These techniques are typically not available in a commercial kit form and may require extensive method development. They are more labor intensive and complex than the colorimetric or absorbance-based assays.

In addition, recovery of the protein from and reproducibility of the capture step complicate validation. Despite these disadvantages, the custom two-step quantitation methods are essential in situations requiring protein specicity. These three regulatory regions have combined to produce International Conference on Harmonization ICH guidelines on many common technical regulatory issues such as analytical assay validation, test procedures, and specications.

ICH, as well as common sense, dictates that an analytical method is suitable for its intended application. Accordingly, all or a combination of the described protein assay methods may be required during the development of a protein biopharmaceutical depending on the particular requirement, be it speed, accuracy, or throughput. The latter value is probably more important because a well-characterized production process is of low priority at this early stage of development. The activity of the target will be the yardstick by which its suitability for further development will be determined.

However, the protein assay precision will be superior to a bioassay by a log, hence activity differences do not result from dosing markedly different quantities of the target protein. Once a target protein has been identied and becomes a clinical candidate, drug development begins in earnest. Any combination of speed, throughput, limit of quantitation, or selectivity could be critical for protein assay at a particular process step. As the purication process progresses, protein purity increases.

Clearly, specicity and limit of detection of the assay are critical for this application. Accuracy and speed are of relatively little importance because the primary concern is relative expression levels and not absolute quantitation. One is looking for order of magnitude differences in expression at the early development stage rather than small percentage optimizations. The ELISA is an example of a two-step method capable of separating the target protein from the milieu by binding the protein to a target-specic antibody and quantitating it via a secondary labeled antibody.

A chromatographic method could also be employed here to separate the target protein from the milieu, followed by spectrophotometric detection and quantitation. Clearly, for applications in which the protein to be quantitated is a minor component of a protein mixture, a custom two-step quantitation method is essential. In contrast to the two-step methods, direct absorbance methods offer speed, simplicity, and accuracy. For these reasons, they are favored in the production area. A min assay is virtually invisible in the manufacturing process; a 4-h assay consumes a workday.

In addition to the technical considerations, there are economic aspects to choosing protein assays. The major weakness of direct absorbance methods is that they require the presence of aromatic residues in the protein.

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Thus other biomolecules, such as nucleic acids, that strongly absorb in the UV region can generate erroneous values. In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purication progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods.

Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training.

Once the protein is puried, it will be formulated to produce the drug product. This could be as simple as diluting the protein in phosphate-buffered saline, or as complex as addition of excipients and lyophilization. The mark of. Hence protein assay is the true workhorse for formulation. Loss of recoverable protein is the rst clue to the occurrence of instability such as denaturation, aggregation, precipitation, or surface adsorption. As described earlier, the highly automatable colorimetric and direct absorbance methods are well suited for use at this stage because many buffers, excipients, and protein contents will be screened during accelerated stability testing to arrive at the drug product formulation.

One commonly occurring problem with formulation studies is interference of excipients with the concentration method. Several different options exist to overcome interference, examples of which are protein precipitation, the use of dyes that are unaffected by the interfering agent, and the use of dyes that form protein complexes with absorbance maxima that are different from the absorbance of the interfering agent. All of the commercially available colorimetric methods describe interfering agents in their literature.

Next, the formulated protein, or drug product, needs to be tested for protein content. The requirement here is to have a product-specic method that accurately determines the dose of drug in the container, is capable of supporting the product through pivotal phase III human clinical trials, and can be validated. Ideally, the method used here will be the method anticipated for commercial production. As the drug goes through development, it is usual for formulations, dosage strengths, and even delivery vehicles to change.

Hence a major challenge for the protein assay at this juncture is for it to remain suitable through the development life cycle. It is worthwhile to develop a drug product protein assay to be as robust and rugged as soon as possible to minimize problems with dose strength later. For example, if one employs an absorbance method during the early phases of product development and fails to identify that the drug is highly aggregated, then light scattering will result in an overestimation of the protein concentration and an unknown true dose.

Clearly, this inaccuracy can be corrected at a later point in the development cycle, but one will probably not be able to accurately assign the doses given in earlier studies because the aggregation state of the protein may well have increased over time. The release of a very expensive product relies on the suitability of the method for its application, so the protein assay needs to give predictably precise and accurate results.

Predictability is achieved through validation as described by the ICH guidelines. Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form.

Many protein instabilities result in precipitation of the protein and adsorption to the container.

2. Analytical Techniques

An instability that results in only a modication of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. Specically, it is Amb a 1, the major allergen of short ragweed pollen, linked to multiple immunostimulatory oligonucleotides ISS.

Amb a 1, which is puried from ragweed pollen, is activated with a heterobifunctional cross-linker and linked to ISS to produce AIC. Hence, protein concentration assays are employed from the Amb a 1 extraction step through to determining the strength of the AIC drug product. The range of purities and environments in which these protein assays are performed during the production process requires several different protein concentration methods to be employed. The rst assay to be employed for protein concentration is the Bradford assay, a commercially available colorimetric assay used to quantitate the total extracted protein.

However, at this step of the production process, the protein concentration is used to calculate nal yields and not to make time-dependent or expensive decisions. Hence the nonspecic Bradford assay is ideal. A simpler direct absorbance method is not suitable due to the presence of a nonprotein chromophore in the ragweed extract. The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom twostep method that employs chromatography to separate the Amb a 1 from the other extracted proteins.

The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of puried Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purication steps, and the Bradford assay adequately reects Amb a 1 concentration through the remainder of the process.



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