1、Designation: F3142 16Standard Guide forEvaluation of in vitro Release of Biomolecules fromBiomaterials Scaffolds for TEMPs1This standard is issued under the fixed designation F3142; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, t
2、he year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 To describe general principles of developing and/orusing an in vitro assay to evaluate biomolecule release
3、frombiomaterials scaffolds for TEMPs, with examples from theliterature1.2 The guide will address scaffolds that do not containseeded cells; general principles may still apply but may need tobe modified if cells are part of the TEMPs.1.3 In vitro release assessment of biomolecules from matri-ces is a
4、 valuable tool for screening biomolecule-scaffoldinteractions, as well as characterization, and/or quality control.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafet
5、y concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F2312 Terminology Relating to Tis
6、sue Engineered MedicalProductsF2809 Terminology Relating to Medical and Surgical Mate-rials and DevicesF2902 Guide for Assessment of Absorbable Polymeric Im-plants2.2 United States Pharmacopeia (USP) General Chapters:3USP Injections and Implanted Drug Products(Parenterals)Product Quality TestsUSP Di
7、ssolutionUSP Drug ReleaseUSP Osmolality and OsmolarityUSP The Dissolution Procedure: Development andValidation2.3 European Pharmacopeia (Ph. Eur.):4Ph. Eur. 2.9.3 Dissolution Test for Solid Dosage FormsPh. Eur. 2.9.4 Dissolution Test for Transdermal PatchesPh. Eur. 2.9.25 Dissolution Test for Medica
8、ted ChewingGums2.4 FDA Document:5FDAGuidance for Industry Bioanalytical Method Validation(2001)3. Terminology3.1 The present document uses the definitions of Terminolo-gies F2809 and F2312.4. Significance and Use4.1 The European Pharmacopoeia (Ph. Eur.) as well as theUnited States Pharmacopeia (USP)
9、 describe several dissolutionand drug release setups for tablets, capsules, transdermalpatches and suppositories (USP , USP , Ph. Eur.2.9.3, Ph. Eur. 2.9.4). However, up to this point no pharmaco-poeia standardized in-vitro release test has been established forparenteral dosage forms which provide s
10、ustained drug release,for example, implants.4.2 An appropriately designed in-vitro release test would befavorable in the early stage of development of biomolecule-releasing scaffolds for TEMPs, as well as in quality control,and may help to reduce the number of animal experiments.4.3 Appendix X1 prov
11、ides a tabulated overview of pub-lished in-vitro release studies performed with biomaterialscaffolds loaded with biomolecules.4.4 One goal of in-vitro release studies is to simulate thein-vivo conditions as closely as possible, but with sufficiently1This test method is under the jurisdiction of ASTM
12、 Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Nov. 15, 2016. Published January 2017. DOI: 10.1520/F3142-16.2For referenced ASTM standards, visit the ASTM website,
13、www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from U.S. Pharmacopeial Convention (USP), 12601 TwinbrookPkwy., Rockville, MD 20852-1790, http:/www.usp.or
14、g.4Available from EDQM Council of Europe, 7 alle Kastner, CS 30026, F-67081Strasbourg, France, or visit the website, www.edqm.eu.5Available from Drug Information Branch (HDF-210), Center for Drug Evalu-ation and Research (CDER), 5600 Fishers Lane, Rockville, MD 20857, or visit thewebsite, http:/www.
15、fda.gov/cder/guidance/index.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles
16、 for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1simplifying abstraction. The simplification comprises two gen-eral aspects: the amount of fluid or release medium in contactwith the implant
17、to simulate the physiological environment,and the composition of that release medium.5. Elements of in vitro Release Assays5.1 Sample (biomaterial scaffold loaded with biomo-ecule(s):5.1.1 The sample should be taken from the final product,manufactured by a representative process including packaginga
18、nd sterilization.5.1.2 There are several considerations that will influence theselection of the sample dimensions.5.1.2.1 Vessel size into which the sample can physically fit.5.1.2.2 Method used to generate a representative sample,where needed, for example, by biopsy punch, cutting with ascalpel bla
19、de.5.1.2.3 Load of the biomolecule, and the concentration inthe final setup.5.2 Release Medium:5.2.1 In order to simulate physiologic conditions at theintended implant site, the release medium should be of appro-priate tonicity (osmolality) and pH. Regarding osmolality, theterm is used according to
20、USP and as such is a measureof concentration of real solutes, expressed in osmole per kgof soluent. Isotonic solutions are iso-osmotic relative to wholeblood. Isotonicity is a target attribute for parenteral injectionsper USP .NOTE 1Cells, for example red blood cells, are susceptible to condi-tions
21、that are outside the isotonic range, by showing swelling throughinflux of water through the cell membrane (potentially leading to burst)when in a hypotonic environment, or shrinking through efflux of waterthrough the cell membrane when in a hypertonic environment. RegardingpH, the medium should be b
22、uffered to prevent pH shifts over time due tothe release of the biomolecule, and/or degradation of the matrix. pH shiftscould change the release mechanism or kinetics.5.2.2 One medium used very often in the published litera-ture is phosphate-buffered saline (PBS), pH 7.2 or 7.4, in therange of isoto
23、nicity (approximately 300 mOsm/kg). In thiscase, the buffer system is provided by phosphate salts; thetonicity is typically augmented with sodium chloride.5.2.3 The release medium may be spiked with other com-ponents to make it more similar to serum, for example, bovineserum albumin (BSA), or fetal
24、bovine serum (FBS; multicom-ponent additive). It should be noted that these additives cansubstantially interfere with the detection method (see 5.7). Ifprotein components are added to the medium (BSA, FBS),evaluation of in vitro release will require more specific and/orsophisticated assays (compared
25、 to simple A280 UV detectionfor concentration), as both the biomolecule that is studied, andthe proteinaceous component(s) of the medium may contributeto the detected signal. The same applies if there are severalbiomolecules that can be released from the biomaterial scaf-fold. Further considerations
26、 are described in 5.8 (especially5.8.4 and 5.8.6). It should also be noted that regarding the useof BSA and FBS, it is important to limit the use to a single orlimited number of lots to reduce variability in the assay system,especially for studies that require more than a single iteration.5.2.4 One
27、of the important factors is to determine if thereare any solubility issues for the biomolecule, in order to definethe most appropriate medium. This primary evaluation can beachieved by exposing the biomolecule to the same experimen-tal conditions without the scaffold present, to create a baselinein
28、the candidate medium, and comparing it to a known stableformulation of the biomolecule at the same concentrationlevels. If the concentrations measured in the candidate mediumare substantially the same as in the known stable formulation,it is a good indicator for sufficient solubility and minimumabso
29、rption to surfaces in the system (see also 5.3.5).5.2.5 If the experiment is set up for an extended period oftime to simulate the target period of release in vivo (that is,several days to weeks or months), consider adding azides orother preservatives to maintain the sterility of the medium andasepti
30、c conditions in the setup. Possible impact of any additiveon either the sample or the analytical method should beevaluated.5.2.6 The ratio of the volume of release medium to biomol-ecule load (which may correspond to the volume or size of theconstruct) is important. If there is a requirement to have
31、 sinkconditions in the experiment (for example, to simulate implan-tation sites with high fluid circulation), then sink conditionscan be achieved by using a large volume of release medium atthe beginning of the experiment and extraction of smallaliquots, or frequent exchange of the medium. Per USPde
32、finition, sink conditions can be maintained at about threetimes the volume of a saturated solution, and it has beensuggested that it should remain below 10 % of saturationconcentration in the solution (Reference: USP ). Asindicated in 5.2.4, and 5.8.8, the saturation concentration in thecandidate me
33、dium may need to be experimentally determined.NOTE 2Solubility of biomolecules is dependent on pH, salts, andtemperature, and may vary widely not only between biomolecules but alsofor each biomolecule at different conditions (for example, BSAreported tobe soluble in water at 40 mg/mL; vendor data sh
34、eets for basic fibroblastgrowth factor (bFGF), nerve growth factor (NGF), and bone morphoge-netic protein-2 (BMP-2) recommend reconstitution of lyophilized materialto not less than 100ug/mL. Note that lyophilized material is typicallyformulated and contains some buffer/salt/stabilizers).5.2.7 The ad
35、dition of a protease or other enzyme may beconsidered, if the biomolecule is expected to be tightly boundor incorporated. The presence of the protease or other enzymemay interfere with the assay used to quantify the biomoleculeof interest (see 5.2.3). Also, the susceptibility of the biomol-ecule of
36、interest to the protease or other enzyme must beexperimentally evaluated.5.2.8 5.2.8 In cases where the biomolecule is tightly boundor incorporated, harsher extraction media may be required. Forexample, for extraction of BMP-2 from bone matrix, bufferscontaining urea (Urist 1984 (1)6, ethylene glyco
37、l (Urist 1979(2), or an arginine-histidine combination (Hollinger 1996 (3)have been described.5.3 Vessel/Container:5.3.1 The vessel needs to be of appropriate size to containthe sample as well as the medium.6The boldface numbers in parentheses refer to a list of references at the end ofthis standard
38、.F3142 1625.3.2 The vessel needs to have a tight closure, and tightconnections in any fluid paths, to keep the system intact. Themain concerns are microbiological contamination, but alsoevaporation which could impact concentration.5.3.3 USP and USP describe several vesselconfigurations that may be a
39、ppropriate and commerciallyavailable to evaluate in vitro release of biomolecules frombiomaterials scaffolds for TEMPs.5.3.4 Vessels may be flow-through cells, or static vesselswith internal, external, or no mixing mechanism. Mixingversus static conditions are discussed further in 5.5.5.3.5 The surf
40、ace and contact materials should be evaluatedregarding adsorption or absorption characteristics of thebiomolecule at the concentrations expected in the experiment.Adsorption/absorption phenomena could have an effect onconcentration results, as well as the structural integrity of themolecule.5.4 Temp
41、erature-Controlled Environment:5.4.1 The experimental condition most often used is 37C,which corresponds to commonly accepted human core tem-perature. Other temperatures may be appropriate for specificapplications or clinical conditions.5.4.2 Temperature-controlled configurations described inUSP and
42、 USP may be appropriate and arecommercially available. Alternatively, appropriate vessels maybe placed in incubators or a water bath. Wrap-around tempera-ture jackets may be appropriate for larger vessels.5.4.3 Increasing the temperature to accelerate release mayor may not be appropriate, as the tem
43、perature may change thecharacteristics or impact the integrity of the scaffold, and thusalter the release mechanism (see Guide F2902). Temperaturealso may impact the integrity of the biomolecule.5.5 Static Conditions versus Dynamic Conditions:5.5.1 The dissolution methods described in the USP areadd
44、ing a stirring movement or flow-through to facilitatetransport and distribution from the surface and immediatevicinity of the dosage form. A static system in vitro wouldlikely also not be reflective of the in vivo situation for TEMPs,although it might be difficult to define a universal fluid flowsim
45、ulating all anatomic sites and conditions.5.5.2 The physiologic condition at the most likely implan-tation scenario should be considered, for example, swellingafter surgery with considerable influx of fluid.5.5.3 Ph. Eur. 2.9.25, dissolution test for medicated chewinggums adds a mechanical stress to
46、 simulate the chewing motion.While the standard frequency (described as 60 per minute) maynot be completely appropriate for TEMPs, the general principleand apparatus might be worth considering in the case where theimplantation site, and thus the TEMPs construct, may beexposed to muscle movement.5.6
47、Sampling Mechanism:5.6.1 Options of pulling samples may be linked to or evendefined by the vessel and general experimental setup, as well asthe assays conducted.5.6.2 In general, the sample pulled may require someseparation of the TEMPs from the release medium. This can beachieved by sampling from t
48、he surface or a location remotefrom the construct to avoid accidentally removing parts of theconstruct. Alternatively, separation of the releasing constructfrom the bulk of the medium has been achieved by placing theconstruct inside a dialysis membrane, or reverse dialysis(construct in the bulk of t
49、he medium, samples pulled from thedialysis cassette).5.6.3 Options include manual sampling, or automation.Automation may decrease the risk of accidental contaminationduring sample pull, as well as operator errors (for example,pulled versus replaced volume, timing, etc.). Some setups mayoffer the option of in-line measurements in a circulatingsystem.5.7 Sampling Time Points and Total Time:5.7.1 The frequency of sampling can influence the releaseby creating sink conditions versus no-sink conditions. Asmentioned above, sink conditions are maintained when thevolume exceeds ap
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