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`Michael 1. Akers et al.
`
`development scientist must keep in mind that the scale of the process may
`influence results. For example, it is commonly assumed that development
`of the freeze-drying cycle changes greatly on going from lab scale to pilot
`scale to production scale. Ideally, experiments can be performed at pilot
`scale using equipment representative of the production process. In
`addition, the sequence of steps in the process, acceptable environmental
`conditions (temperature, humidity, air, etc.) for the unit operation, and
`acceptable excipient ranges should be evaluated.
`For solutions, the following process conditions may impact product
`quality:
`
`1. Agitation (rate, duration): High rates may result in undesired
`foaming, denaturation, aggregation, or oxidation. Kim et al. (1994)
`reported on the effect of high shear force on the IX helix-to-fJ sheet
`conversion of insulinotropin resulting in a reduced solubility of the
`protein. Thus, agitation or mixing rates will affect the conformation
`and solubility of this protein, thus requiring special control of these
`rates.
`2. Compounding sequence: Order of addition of excipients and drug
`substance must be determined. Normally, excipients are added and
`dissolved before drug substance is added (Harwood et al., 1993).
`This allows sufficient time for excipients to dissolve while minimiz(cid:173)
`ing time in solution for unstable drug substances.
`3. pH adjustments: Prolonged exposure to acid or basic pHs can cause
`protein degradation.
`4. Filtration: A membrane is selected which offers low protein binding
`as confirmed by protein assays on the solutions before and after
`filtration to detect any losses (Hawker and Hawker, 1975). This is
`discussed in detail in Chapter 5.
`5. Filtration and filling: The temperature may need to be controlled to
`prevent chemical degradation during filling. Rates of filtration and
`filling should be considered to prevent shearing of the protein,
`although this possibility is remote. Effects of filtration and filling
`rates were studied for human growth hormone (Hsu et al., 1988;
`Pikal et al., 1991 b). Shear forces encountered during processing were
`found not to cause aggregation of human growth hormone.
`However, aggregation of proteins during filtration and filling can
`be caused by interactions of the protein with polymeric hydrophobic
`surfaces, such as those composing sterilizing filters and process
`tubing for filling equipment, suspected to be the cause of human
`growth hormone aggregation during processing (Hsu et aI., 1988).
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`
`Insulin aggregation and fibrillation can be caused by interaction of
`the protein in solution with plastics (Thurow and Geisen, 1984) (see
`previous discussion on insulin binding to plastic surfaces).
`6. Freeze-drying: Denaturation of a protein solution may occur
`because of pH shifts or ionic strength changes during freezing (Orii
`and Morita, 1977). The freezing rate, concentration, endpoint pH,
`and time and temperature of holding prior to lyophilization may
`affect the chemical and physical stability of the product (see Chapter
`6).
`7. Environmental conditions: Sparging of nitrogen into solution to
`remove dissolved gases or use of a nitrogen overlay to replace the air
`headspace during filling to retard the oxidation of oxygen-labile
`formulation may be required (Brown and Leeson, 1969).
`8. Materials compatibility: During the manufacture, the drug is
`exposed to various materials such as stainless steel, filters, tubing,
`and pump diaphragms. It is
`important to ensure
`that the
`formulation components are compatible with these materials.
`9. Time and temperature: Throughout manufacture, critical holding
`times and temperatures need to be established, not only for protein
`stability protection purposes, but also to assure microbiological
`control, particularly to prevent endotoxin contamination.
`
`See Chapter 7 on quality assurance and quality control for additional
`details on this topic.
`
`12.3. Clinical Trial Supplies
`
`As the transfer of a process from laboratory scale into the clinical
`phase occurs, the development scientist needs to be involved during the
`early production of clinical trial supplies as well as during any subsequent
`formulation and process changes. The development scientist can obtain
`valuable insight into the conditions of manufacturing as well as provide
`key
`input
`to
`the manufacturing groups about
`the
`rationale for
`formulation and process decisions. These lots also provide an opportunity
`to collect information under larger scale manufacturing conditions than
`in laboratory-scale experiments. In-process testing should be routinely
`conducted during the manufacture of clinical supplies. The data are
`used to evaluate the process and lead to process improvements, which
`ultimately support the transfer into the manufacturing phase at full
`scale.
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`13. QUALITY CONSIDERATIONS DURING FORMULATION
`DEVELOPMENT
`
`13.1. Early Product Assessment
`
`All known physical and chemical properties of a protein should be
`viewed in light of how these properties will affect final quality of the
`finished dosage form. The development scientist should build quality into
`the process so that it can be validated and data exist to support "worst(cid:173)
`case" limits (e.g., extremes in pH, excipient levels, time/temperature limits).
`Process design should be evaluated to assure control of quality parameters.
`
`13.2. Documentation
`
`An essential element of validation is establishing thorough documenta(cid:173)
`tion that a process will consistently meet its predetermined specifications
`and quality attributes. Ongoing documentation is essential during formula(cid:173)
`tion development. Development history reports are required during
`preapproval inspections and are generally a high-level overview describing
`the history of the drug product from preliminary studies to the commercial
`formulation and the process submitted in the regulatory document. There
`may be one report or several to describe the various steps of development
`(e.g., fermentation, granule isolation, drug substance, drug product,
`methods development). It is as important to document what did not work
`as what worked and why decisions were made. Where necessary, bioequi(cid:173)
`valence of lots used in clinical trials should be demonstrated. A method
`history report should contain information on regulatory commitments
`during the development process and give the history of each method.
`Rationale for specifications, reference standards, and cleaning methods
`should be provided. For product development reports, lot rejections,
`deviations, and resolution of the issues need to be covered. Typical contents
`of a parenteral drug product development history report include the
`following:
`
`• Preformulation data
`• Selection of excipients supported by preformulation studies
`• Antimicrobial characteristics of the product
`• Rationale and basis for packaging component choices
`• Description of container/closure integrity studies
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`• Overview of the manufacturing process with a description of each
`unit operation, acceptance criteria for all critical steps of the
`process, and a history of results for each step
`• Scale-up studies
`• Stability overview, including product use studies
`
`Development history reports include, but are not limited to, method
`histories, formulation, primary packaging, process development, and control
`strategy.
`Technical reports prepared on an ongoing basis aid in compiling the
`development history reports and are key in the information transfer to the
`manufacturing sites. Typical information contained in these periodic reports
`are the rationale for decisions, description of what worked and what did not
`work, and information for solving manufacturing problems.
`
`13.3. Stability Studies
`
`Stability testing of protein and peptide dosage forms should follow
`the ICH guidelines (U.S. FDA, 1996; ICH, 1995). Table XVI summarizes
`the requirements of this guideline. Three batches or more of the final
`product in the final container/closure system that represents the finished
`product manufacturing scale must be put on stability testing. The batches
`should use different lots of bulk material. A minimum of 6 months of data
`at desired storage conditions must be available at the time of submission
`(less than 6 months of data for products that will have less than 6 months
`dating). Product expiration dating
`is based on real-time data, not
`extrapolated from accelerated stability studies. If different volumes and/
`or strengths of the same formulation are to be tested, a matrix system or
`bracketing may be permitted. Details of matrixing and bracketing are
`found in the guideline.
`Stability testing must use methods that are stability-indicating and
`validated. Methods should monitor changes in potency, purity, and other
`product characteristics* as a function of time and storage conditions as
`defined in the stability protocol. Data on final .product in containers
`maintained in an inverted or horizontal position and all different container/
`closure combinations must be obtained. Multiple dose containers must have
`data to support stability during simulated use, for example, repeated
`
`·Visual appearance, visible particulates in solutions. pH, moisture level of powders, sterility
`testing or container/closure integrity, degradation, if any. of additives.
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`Table XVI
`Summary of Guideline for Stability Testing of Biotechnological and Biological
`Productsa
`
`Selection of batches
`
`Guidance for stability studies for regulatory submissions
`
`A. Drug substance
`
`B. Intermediates
`
`C. Drug product
`
`D. Sample selection
`
`Stability-indicating
`profile
`
`A. Protocol
`
`B. Potency
`
`C. Purity/molecular
`characteristics
`
`At least three batches representative of production scale
`A minimum of 6 months of stability data at time of submission (unless
`storage period will be less than 6 months)
`Pilot-plant-scale batch data acceptable at submission as long as there is
`commitment to place first three manufacturing-scale batches into
`long-term stability program after approval
`Storage containers should represent actual holding containers to be
`used during manufacture; containers of reduced size acceptable,
`provided they are constructed of same material and use same type
`of container/closure system
`Identify intermediates and generate in-house data and process limits to
`assure final product stability
`At least three batches of final container product representative of final
`product at production scale
`Different batches of bulk material should be studied in final product
`A minimum of 6 months of data at time of submission (unless storage
`period will be less than 6 months)
`Product expiration dating based on real-time/real-temperature data
`Continuing stability updates should occur during review process
`Quality of final product must be representative of quality of material
`used in clinical studies
`Pilot-plant-scale batch data acceptable at submission as long as there is
`commitment to place first three manufacturing scale batches into
`long-term stability program after approval
`Matrix system and/or bracketing is acceptable when product consists
`of different fill volumes, units, or mass
`
`Guidance
`
`Include detailed protocol for assessment of stability of both bulk drug
`substance and drug product to support proposed storage conditions
`and expiration dating periods; include statistical methods, specifica(cid:173)
`tions, test intervals
`Potency must be compared to an appropriate reference standard
`Perform at appropriate intervals as defined in the protocol and report
`in units of biological activity calibrated against some recognized
`standard
`Purity should be assessed by more than one method
`Limits of acceptable degradation should be documented and justified,
`taking into account levels observed in material used in preclinical and
`clinical studies
`Usual methods include electrophoresis, high-resolution chromatrogra(cid:173)
`phy, and peptide mapping
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`
`Stability-indicating
`profile
`
`D. Other product
`characteristics
`
`Storage conditions
`
`A. Temperature
`
`B. Humidity
`
`C. Accelerated and
`stress conditions
`
`D. Light
`
`E. Container/closure
`
`F. Stability after
`reconstitution of
`freeze-dried product
`Testing frequency
`:;;:; I year
`Shelf life
`Shelf life ~ I year
`
`Table XVI
`(continued)
`
`Guidance
`
`Visual appearance, visible particulates in ready-to-use or reconstituted
`solutions, pH, moisture level of solid products
`Sterility testing or alternatives (e.g., container/closure integrity) should
`be performed at a minimum initially and at the end of the proposed
`shelf life
`Additives such as stabilizers and preservatives that may degrade
`during the dating period of the product and might adversely
`affect product quality need to be monitored during stability
`
`Guidance
`
`Storage conditions for real-time/real-temperature stability studies may
`be confined to the proposed storage temperature
`As long as humidity-protected containers are used, stability tests at
`different relative humidities can be omitted
`Studies should be conducted on the drug substance and drug product
`under accelerated and stress conditions to provide useful support
`data for establishing the expiration data, be used for support of
`change in formulation or scaleup, assist in analytical method
`validation, or generate information that may help elucidate
`degradation profile of the drug; other benefits include data on
`accidental exposure to stress conditions, e.g., during transportation,
`determine what test parameters are best indicators of product
`stability, and give further insight into degradation patterns not seen
`under normal storage conditions
`Appropriate regulatory authories should be consulted on a case-by(cid:173)
`case basis
`Stability studies should include samples maintained in inverted or
`horizontal position for product to have maximum contact with
`closure
`Data should be supplied for all different container/closure combina(cid:173)
`tions that will be marketed
`Data must be generated that demonstrate that the closure used for
`multiple-dose application is capable of withstanding repeated
`insertions and withdrawals so that product retains full potency,
`purity, and quality
`Stability of product after reconstitution should be demonstrated for
`the conditions and maximum storage period specified
`
`Monthly testing for first 3 months; 3-month intervals thereafter
`Every 3 months during first year of storage, every 6 months during
`second year; annually thereafter
`
`"Summarized from FDA (1996). Also published in Federal Register on July 10, 1996 (61 FR 36466).
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`Michael J. Akers et at.
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`punctures of the closure, effect of volume change due to repeated
`withdrawals on potency, purity, and quality.
`Stability testing intervals usually are every 3 months during the first
`year of storage, every 6 months during the second year, and annually
`thereafter.
`Although accelerated stability testing is not acceptable for direct
`assignment and support of expiration dates for finished protein products, it
`does have a role in developmental stability studies. ICH stability guidelines
`emphasize the importance of conducting stability studies under accelerated
`conditions, because such studies usually provide useful supporting data for
`establishing the expiration date. Additionally, accelerated stability studies
`provide information for short-term assessment of proposed formulation,
`packaging, or manufacturing changes, evaluate equivalence of materials
`from different bulk process methods, assist in the validation of analytical
`methods for the stability program, and can generate information which may
`help elucidate degradation profiles of the protein alone and in the final
`formulation.
`Accelerated stability studies also may be useful in determining whether
`accidental exposures to stress conditions during transportation of the final
`product are harmful to the product. The guidelines generally define
`accelerated stability test conditions at temperatures at least 15°C above
`the designated long-term storage temperature for the product.
`Typically, kinetic experiments are conducted at elevated temperatures.
`Estimates of rate constants at lower temperatures are then obtained by
`extrapolation of an Arrhenius plot. An assumption of this approach is that
`the kinetics does not change as a function of temperature. For proteins, it is
`important to determine the appropriate temperature range for these stability
`studies (Yoshioka et al., 1994). Ideally, it is desirable to obtain an
`approximately linear relationship to permit prediction of shelf life of the
`drug product. However, in cases where the results cannot be extrapolated,
`these data are still useful for determining the effects of short-term
`temperature excursions on the product and for specifying any special storage
`precautions that may be encountered during shipping and distribution.
`Shnek et al. (1998) described two automated physical stress tests for
`evaluating physical stability of insulin suspensions and solutions. One is a
`moderate stress test combining temperature cycling (25°C to 37°C) and
`resuspenion by agitation, and the other is a more considerable stress test
`combining high temperature (37°C) and extreme agitation (4 h daily). These
`tests provide more relevant physical stability predictions for using
`suspensions and solutions in pen-cartridge devices and determining effects
`of stressful conditions these protein products could encounter during
`shipping, distribution, and patient use.
`
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`111
`
`13.4. Stability Studies Supporting Distribution of Protein Products
`
`Formulation development typically focuses on protein dosage form
`stability under well-controlled conditions. However, studies must be done to
`demonstrate that the final protein formulation in the final package produced by
`a validated process will retain its stability and other quality properties during
`distribution of the product throughout the world. These studies can be
`accomplished both by simulation in the laboratory and actual distribution of
`the product. During distribution, even if the product is to be held in controlled
`(e.g., refrigerated) conditions, aberrant situations can occur, such as due to
`transportation breakdown, mechanical failure, dropping or otherwise mis(cid:173)
`handling packages, and even overt violation of required handling procedures.
`Data should be available to aid in knowing what these extreme situations will do
`to the quality of the product. The effect of shear (i.e., agitation, mixing, and
`other mechanical forces experienced during processing and handling) and
`temperature extremes on protein stability during simulated and actual
`conditions should be evaluated.
`
`14. EXAMPLES OF FORMULATION PROBLEMS
`
`14.1. Aggregation
`
`Protein aggregation, as already emphasized in this chapter, is a major
`problem in developing stable protein solutions. * Acidic fibroblast growth
`factor (aFGF) is an example of a protein that aggregates readily when
`exposed to temperatures above refrigeration (Tsai et aI., 1993). To solve this
`problem, the authors described a four-part approach:
`
`1. Use a rapid screening procedure, based on turbidimetric measure(cid:173)
`ment, to identify solution conditions, polyanions, and common
`excipients that stabilize aFGF against heat-induced aggregation.
`2. Combine the most promising agents with aFGF and use circular
`dichroism and differential scanning calorimetry to quantitate
`stabilization effects against aggregation.
`3. Formulate the most promising agents with aFGF in solution and
`monitor protein stability at room temperature.
`
`*For more thorough treatment of protein formulation problems, the reader is referred to the
`two books edited by Wang and Pearlman (1993; Pearlman and Wang, 1996) on case histories
`involving stability and characterization of protein and peptide drugs.
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`Michael J. Akers et al.
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`4. Test in vivo efficacy of the best formulations of aFGF using a wound
`healing animal model.
`
`This approach enabled the authors to learn that in addition to the
`well-known protection of aFGF by heparin, a surprisingly wide variety of
`polyanions (e.g., ATP, dextran sulfate, pentosan polysulfate, sulodexide,
`phytic acid, inositol hexasulfate, inorganic phosphate salts) stabilize aFGF
`against aggregation by increasing the temperature at which the protein
`unfolds by I5-30°C. Stabilization by polyanionic compounds occurs
`because these compounds enhance the structural integrity by binding to
`the protein. Common excipients also were studied for their stabilizing
`effects. Many were effective (e.g., glycine, glycerol, sucrose, dextrose,
`trehalose), but at relatively high concentrations. Their final formulation
`choices, all of which were equally capable of accelerating wound healing,
`contained either heparin, inositol hexasulfate, or sulfated p-cyclodextrin
`(with the latter two formulations also containing 1 % hydroxyethyl
`cellulose in phosphate buffer with 0.2 mM EDT A). This is an excellent
`paper from which to learn approaches for solving protein aggregation
`problems.
`
`14.2. Oxidation and Deamidation
`
`Human growth hormone is a protein that both in aqueous solution and
`in the solid state can undergo chemical decomposition both by oxidation
`(methionine 14 to methionine sulfoxide) and deamidation (asparagine at
`position 149) (Becker et ai., 1987). This protein is also prone to aggregation.
`Pikal et ai. (199Ia, b) studied how to overcome these stability problems.
`Variables that they studied included pH, levels of salts, type of lyoprotectant
`excipient, residual water content, and oxygen level in the vial headspace.
`They found that a combination of glycine and mannitol enhanced stability,
`largely because glycine in this combination can remain amorphous. In the
`amorphous state glycine serves as a lyoprotectant and as a "sink" for
`residual moisture, both effects serving to stabilize the protein against
`deamidation and aggregation. The level of phosphate buffer is important as
`an aid in the minimization of deamidation, as a pH value on either side of
`pH 7 increased chemical decomposition. Increases in levels of sodium
`chloride increased the rate of decomposition.
`Oxidation of human growth hormone is due to methionine oxidation.
`Oxidation occurred even when the air in the vial headspace was replaced
`with nitrogen (headspace < 1 % oxygen). Phosphate-buffered formulations
`containing glycine and mannitol as stabilizing excipients, although found to
`
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`Formulation Development of Protein Dosage Forms
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`
`be overall the most stable of all hGH formulations studied (Pikal et ai.,
`1991a), were also found to be the most sensitive to changes in the oxygen
`level of the vial headspace. Because these formulations are amorphous due
`to the effect of glycine in the presence of mannitol, any dissolution of
`headspace oxygen in the amorphous phase can significantly affect protein
`oxidation. It was assumed by the authors that most of the oxygen supply in
`the formulated vial with nitrogen headspace came from oxygen "trapped" in
`the amorphous phase of the product during freeze-drying, the trapped
`oxygen originally being dissolved in the solution filled into the vial.
`
`14.3. Binding to Glass
`
`Early identification of the properties of surface adsorption to solid
`surfaces such as glass is critical to the development of packaging and
`delivery systems for proteins. As discussed in earlier sections, various
`formulation additives can be added to minimize or inhibit adsorption.
`Johnston (1996) examined the effects of solvent additives which would
`minimize the degree of adsorption to various surfaces for a model protein,
`recombinant human granulocyte colony stimulating factor (rGH-CSF). For
`glass vials, he examined solutions containing the following additives: 0.5%
`w/w PluronicC!t) F-l27, 0.05% and 0.5% w/w PluronicCR) F-68, 0.5% v/v
`glycerin USP, and 0.005%, 0.05%, and 0.5% Tween 20. A concentration of
`
`Table XVII
`Adsorption of Bovine Serum Albumin (BSA) and Bovine Immunoglobulin G (IgG)
`to Commercial O.2-llm Microfiltration Capsuleso
`
`Membrane type and
`capsule manufacturer
`
`Protein concentration
`Ilg/ml; (wt%)
`
`BSA adsorption
`Ilg/cm2
`
`IgG adsorption
`Ilg/cm2
`
`Polyvinylidene difluoride,
`millipore
`
`Nylon, Pall
`
`Cellulose acetate, sartorius
`
`50 (0.0050)
`250 (0.025)
`1000 (0.10)
`5000 (0.50)
`50 (0.0050)
`250 (0.025)
`1000 (0.10)
`5000 (0.50)
`50 (0.0050)
`250 (0.025)
`1000 (0.10)
`5000 (0.50)
`
`2
`11
`56
`6
`31
`31
`102
`
`5
`17
`56
`
`1
`3
`
`108
`126
`
`1
`5
`
`"Reproduced with permission from Brose and Waibel (1996). Copyright 1996 Aster Publishing.
`
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`2 ng/ml rGH-CSF in DSW was used as a control. Each solvent additive was
`examined in triplicate at each concentration tested, and solutions were
`sampled at times 0, O.S, 1, 2, and 3 hr and assayed for rGH-CSF. A rapid
`adsorption was observed up to 1 hr, after which time a slower rate
`approaching steady state was observed, similar to adsorption reported for
`insulins and other proteins. Furthermore, viscometry was applied to
`estimate that the thickness of the adsorbed layer to glass was about I !lm.
`Tween 20 at a concentration of O.S% showed the most potential for
`inhibiting surface adsorption to parenteral glass vials.
`
`14.4. Binding to Filter Surfaces
`
`Proteins are known to bind to filter surfaces. Brose and Waibel (1996)
`reported on the adsorption of BSA and sheep IgG to three types of commercial
`filter surfaces, polyvinylidene difluoride (PVDF, Millipore), nylon (Ultipor
`N66, Pall), and cellulose acetate (CA, Sartorius). They filtered S-mg/ml
`solutions of proteins at pressures < 1 psi and measured protein concentration
`in the filtrate as a function of filtrate mass. The mass of adsorbed protein was
`determined by measuring the difference between theoretical (S mg/ml = O.S
`wt%) and actual protein weight percent. This mass was divided by the
`membrane surface area (SOO cm2) to obtain the amount of protein adsorbed
`per unit surface area. They found that both proteins adsorb to all filters, but
`the extent of adsorption is markedly affected by protein concentration and the
`type of filter (see Table XVII). Nylon filters adsorbed the most protein. BSA
`adsorption was linearly related to its concentration, with adsorption to nylon
`being twice as high as its adsorption to PVDF and CA membranes. IgG
`adsorption was linear, but very low to PVDF and CA filters, whereas its
`adsorption to nylon was extensive and nonlinear (it leveled off at higher
`concentrations). Other authors (Brose and Waibel, 1996) have also reported
`on protein adsorption on filter surfaces, so the phenomenon will occur, but the
`question is how much and how can the adsorption be minimized? The choice
`of filter, the concentration and type of protein being filtered, and the filtration
`conditions (pressure, rate, size of filter, etc.) all will affect the extent of protein
`adsorption to filters.
`
`REFERENCES
`
`Absolom, D. R., Zingg, W., and Neumann, A. W., 1987, Protein adsorption to
`polymer particles: Role of surface properties,J. Biomed. Mater. Res. 21:161-171.
`
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