A Novel in Vitro Delivery System for Assessing the Biological Integrity of Protein upon Release from PLGA Microspheres (original) (raw)
Preparation and in vitro release profiling of PLGA microspheres containing BSA as a model protein
Brazilian Journal of Pharmaceutical Sciences, 2020
Conventional drug formulations are incapable of adequate delivery of proteins and peptides for therapeutic purposes. As these molecules have very short biological half-life, multiple dosing is required to achieve the desirable therapeutic effects. Microspheres are able to encapsulate proteins and peptide in the polymeric matrix while protecting them from enzymatic degradation. In this study Bovine Serum Albumin (BSA) matrix type microspheres were fabricated using Polylactideco-glycolide (PLGA) by double emulsion solvent evaporation method. The effects of variables such as homogenizer speed, molecular weight of polymer and the effect of pH of the water phases, were investigated against factors such as drug loading, encapsulation efficiency, morphology, size, drug distribution and release profile of the microspheres. Results, suggested that an increase in homogenization speed leads to a decrease in microsphere size. The increase in homogenization speed also caused a significant effect on the release profile only when higher molecular weight of polymer had been used.. The pH change of the internal aqueous phase led to modification of surface morphology of spheres to a porous structure that significantly increased the total amount of released protein. Integrity of protein structure was intact as shown by SDS-PAGE. According to the results, it can be concluded that we achieved a reproducible method regarding controlled protein delivery for different sizes of particles.
Journal of Controlled Release, 1991
Poly(o~-lactide/glycolide, 50: 50) microspheres containing bovine serum albumin (RSA) were prepared with and without Carbopol" 95 I (a potential adjuvant agent) by o/o, o/w and (w/o)lw emulsion methods. The protein loading of the microspheres reached 50-700/o of the theoretical amount of protein put into the formulation medium. The microsphere particle size was approximately 500 pm, 2%IOOpm, IO-20pm usingojo, o/w, or (w/o)/ w emulsion techniques. respectively. The release of BSA was dependent on the preparation method. The greatest burst of release was found for vacuumdried microspheres formulated using the (w/0)/w method. This burst effect could be eliminated by lyophilizing the microspheres following their preparation. BSA was released at a higher initial rate from microspheres prepared by the o/w emulsion method that contained Carbopol" 95 I than from microspheres not containing CarbopolO 951. Release studies also suggested that the release of SSA could be sustained for 54, 36, or 34 days pa! microspheres prepared by o/o. o/w, or (w/o)@ methods, respectively. I 2 3 4 5 6 7 8 9 10 efemnces R.J. Linhardt, Bvadegradable polymers for the controlled release ofdrugs. In: M. Roroff (Ed. ), Controlled Releaseof Drugs: Po,ymersand Aggrcgatc Systems. VCH Publishers, New York, 1989, pp. 53-95. K. Junl and M. Nakano, Poly(hydroxy acids) in drug delivery. Crit. Rev. Ther. Drug Carrier Syst., 3 (1987) X9-232. J.P. Kilchell and D.L. Wise, Poly(lactide/glycolide) biodegradable drug-polymer matrix system. In: K.J. Widderand R. Green (Eds.), Methods in Enzymology, Vol. 112. Academic Press, Orlando, FL, 1985, pp. 436-448. J. Hcller. Zero order drug release from biocrodibk polp men. In: J.M. Anderson and S.W. Kim (Eds.), Recent Advancer in Drug Dclivcv Systems, Plenum Press New York. 1984,~~. 101-121. T.R. Tice and D.R. Cowsar. Biodegradable mntrolledrclcasc parenteral systems, Pharm. Tech., 8( I I ) ( ,984, 26-35. R.S. Langcr and N.A. Pcppas. Prcscnt and future applicauons of biomatcrials in controlled drug delivery systems. Biomalerials, 2 (1981 ) 201-214. D.A. Wood, Biodegmdable drug d&very systems, Jnt. J. Phsrm..7 (1980, l-18. S. Yolbs and M.F. Sanon, Dcgradablc polymers for sustained drug release. In: R.L. Juliano (Ed. ). Drug DP livery Systems, Oxford University Press, New York, ,980, pp. 84-1 IO. A. Schindler, R. Jeffcoat,G.L. Kimmel. C.G. Pit1.M.E. Wall and R. Zweldmger. Biodegradable polymers for sustained drug delivery. In: E.M. Pearce and J.R. Schaeffgrn (Eds ). Conrcmporary Topics in Polymer scwnce. "cd. 2. Acnum Press, New York, 1977, pp. 25 I-286. N. Marcotlc, A. Polk and M.F.A. Gooaen. Kinetics of protcm release from a poly(w-lactide) reservoir systcm.J.Pharm.Sci.. 79(5) (1990) 407-410. 26 R.J Llnhaidt, DR. Fhagim. E. SEhrnl,, and H.T. Wang. Blodcgradable poly(eners, and [he debvwy of bloacwc Bg",s. Pobm. Prcpr., 31(,) (1990, x9-259. ConwAled release of prowm and vatcmes from poly(e9er) microsphcrcs in vitro. In: G. 00. b&in (Ed 1. Polymers for Cosmeuc and Pharmaceuucal Appbcauons. Plenum, New York, I991 ~ in press. 29 J Kreuler and E. Liehl. Long-term studies of nucroenprolidc accLatc into microcapwles of polylactlc or copoly(lacuc,Slycol,c, acid. Cbem. Pbarm. Bull.. 36( 3, (1988) 1095-1103 35 M. Bradford, A raped and scnsilw method for the quanl~tation of microgram qu3nIilies of protein utiliring the principle of pmlem-dye bindtng. Anal. Biochem.. 72 (1976) 248-254. 36 G.L. Gualandi. NM Losio. C. Muratori and E. Font. The ab~bty by dxfferem preparations of porcine parve-+I~P to enhance humoral immunity m swine and guinea p,gs, MIcroblolag,ca. I I t 1988) 363-369.
Effect of additives on the release of a model protein from PLGA microspheres
AAPS PharmSciTech, 2001
The purpose of this study was to investigate the effect of 2 additives, poly(ethylene glycol) (PEG) 1000 and 1,2,3-tridecanoyl glycerol (tricaprin), on the physico-chemical characteristics and in vitro release of a model protein, bovine serum albumin (BSA), form poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. BSA-loaded microspheres were prepared by the double emulsion solvent evaporation method. Additives were incorporated into microspheres to modify the release of protein. The addition of PEG 1000 and tricaprin changed the surface characteristics of microspheres from smooth and nonporous to porous and dimpled, respectively. The in vitro release profiles showed that the additives significantly (P < 0.05) increased the early-stage release of BSA from microspheres.
European Journal of Pharmaceutics and Biopharmaceutics, 2010
Incomplete protein release from PLGA-based microspheres due to protein interactions with the polymer is one of the main issues in the development of PLGA protein-loaded microspheres. In this study, a twodimensional adsorption model was designed to rapidly assess the anti-adsorption effect of formulation components (additives, additives blended with the polymer or modified polymers). Lysozyme was chosen as a model protein because of its strong, non-specific adsorption on the PLGA surface. This study showed that PEGs, poloxamer 188 and BSA totally inhibited protein adsorption onto the PLGA37.5/25 layer. Similarly, it was emphasised that more hydrophilic polymers were less prone to protein adsorption. The correlation between this model and the in vitro release profile was made by microencapsulating lysozyme with a low loading in the presence of these excipients by a non-denaturing s/o/w encapsulation technique. The precipitation of lysozyme with the amphiphilic poloxamer 188 prior to encapsulation exhibited continuous release of active lysozyme over 3 weeks without any burst effect. To promote lysozyme release in the latter stage of release, a PLGA-PEG-PLGA tribloc copolymer was used; lysozyme was continuously released over 45 days in a biologically active form.
Controlled Release of Lysozyme from Double-Walled Poly(Lactide-Co-Glycolide) (PLGA) Microspheres
Polymers
Double-walled microspheres based on poly(lactide-co-glycolide) (PLGA) are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w 1 /o/o/w 2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52 ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose) showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose) provide a controlled and sustained release for lysozyme.
International Journal of Molecular Sciences, 2021
Over the past few decades, long acting injectable (LAI) depots of polylactide-co-glycolide (PLGA) or polylactic acid (PLA) based microspheres have been developed for controlled drug delivery to reduce dosing frequency and to improve the therapeutic effects. Biopharmaceuticals such as proteins and peptides are encapsulated in the microspheres to increase their bioavailability and provide a long release period (days or months) with constant drug plasma concentration. The biodegradable and biocompatible properties of PLGA/PLA polymers, including but not limited to molecular weight, end group, lactide to glycolide ratio, and minor manufacturing changes, could greatly affect the quality attributes of microsphere formulations such as release profile, size, encapsulation efficiency, and bioactivity of biopharmaceuticals. Besides, the encapsulated proteins/peptides are susceptible to harsh processing conditions associated with microsphere fabrication methods, including exposure to organic s...
The AAPS Journal, 2009
The reduced injection frequency and more nearly constant serum concentrations afforded by sustained release devices have been exploited for the chronic delivery of several therapeutic peptides via poly(lactide-co-glycolide) (PLG) microspheres. The clinical success of these formulations has motivated the exploration of similar depot systems for chronic protein delivery; however, this application has not been fully realized in practice. Problems with the delivery of unmodified proteins in PLG depot systems include high initial "burst" release and irreversible adsorption of protein to the biodegradable polymer. Further, protein activity may be lost due to the damaging effects of protein-interface and protein-surface interactions that occur during both microsphere formation and release. Several techniques are discussed in this review that may improve the performance of PLG depot delivery systems for proteins. One promising approach is the covalent attachment of poly(ethylene glycol) (PEG) to the protein prior to encapsulation in the PLG microspheres. The combination of the extended circulation time of PEGylated proteins and the shielding and potential stabilizing effects of the attached PEG may lead to improved release kinetics from PLG microsphere system and more complete release of the active conjugate.
Development of polylactide microspheres for protein encapsulation and delivery
Journal of Applied Polymer Science, 2002
The development of injectable microparticles for protein delivery is a major challenge. We demonstrated the possibility of entrapping human serum albumin (HSA) and thrombin (Thr) in poly(ethylene glycol) (PEG)-coated, monodisperse, biodegradable microspheres with a mean diameter of about 10 m. In our earlier studies, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) analysis was used to characterize the surface of PEG-coated, taxol-loaded poly(lactic acid) (PLA) microspheres. An analysis by DRIFTS revealed that PEG was incorporated well on the PLA microsphere surface. An emulsion of protein (in water) and PLA dissolved in an acetone-dichloromethane (or acetone-chloroform) mixture were poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA)] with stirring with a high-speed homogenizer for the formation of microparticles. HSA recovery in microspheres ranged from 13 to 40%, depending on the solvent and emulsification systems used for the preparation. PLA dissolved in a dichloromethane/acetone system and albumin loaded via a PEG emulsification solution (PLA-PEG-HSA) showed maximum drug recovery (39.5%) and drug content (9.9%). Scanning electron microscopy revealed that PEG-coated microspheres had less surface micropores than PVA-based preparations. The drug-release behavior of microspheres suspended in phosphate-buffered saline exhibited a biphasic pattern. An initial burst release (30%) followed by a constant slow release for 20 days was observed for HSA and Thr from PLA-PEG microspheres. PEG-coated PLA microspheres show great potential for protein-based drug delivery.
A potential approach for decreasing the burst effect of protein from PLGA microspheres
Journal of Pharmaceutical Sciences, 2003
A central issue in controlled delivery of therapeutics from biodegradable microspheres is the immediate burst of drug release upon injection. This burst is often observed with microsphere systems made by the double emulsion (w/o/w) technique, and may be prevented by improving the drug distribution throughout the polymer matrix. To this end, protein and polymer (poly-lactide-co-glycolide or PLGA) were dissolved within the same solvent system, and micron-sized microspheres were created from this solution by spontaneous emulsification. Improved protein loading was achieved by ion-pairing the protein with charged surfactants to increase solubility in the single-phase solvent system. Both in vitro and in vivo results showed a much diminished burst: compared to microspheres made by double emulsion, it was reduced over 10-fold. ß
Development and Scale-up of a Microsphere Protein Delivery System
Biotechnology Progress, 1998
This paper reviews the development path for the ProLease injectable microsphere delivery system for proteins using human growth hormone as an example. The process consists of four stages, the selection of a lead formulation for clinical testing, the preclinical evaluation of the lead formulation including toxicology and stability studies, the manufacture of phase I clinical supplies, and the scale-up for phase II and phase III clinical trials. The approaches used to overcome obstacles during each stage are summarized including ways of stabilizing the protein, obtaining desirable release kinetics, and manufacturing sterile batches for clinical testing. Stability, release, toxicology, and scale-up results for ProLease recombinant human growth hormone (rhGH) are given. The phase I clinical data show that bioactive rhGH was released for about 1 month in humans. This work shows that processes and procedures have been developed that enable the production of microsphere sustained release formulations for proteins suitable for clinical trails and commercialization.
Journal of Biomedical Materials Research Part A, 2013
Dextrans show great promise for delivery of therapeutic agents. Dextran acetates (DAs) were synthesized with increasing degrees of substitution (DA1 < DA2 < DA3) by the reaction of the polysaccharide dextran (70 kDa) with acetic anhydride. A series of polyethylene glycol (PEG)/DA microspheres were prepared and tested with bovine serum albumin (BSA) functioning as a model protein. Particle size (0.74-0.85 lm) and encapsulation efficiency (56-70%) increased with the degree of substitution along with a slower release rate of protein from PEG/DA microspheres. Time to release 90% of protein rose from 31 to 118 min. Percentage of BSA released from PEG and PEG/DA3 microspheres with time (min) was modeled mathematically [Y PEG ¼ 100(1 À e À0.12t); Y PEG/DA3 ¼ 100(1 À e À0.024t)] to predict cumulative delivery from mixtures in vitro over a period of hours when constrained to a target level at 30 min. The system is examined for potential application in thrombolytic therapy. V
Molecular Pharmaceutics, 2006
A new microencapsulation technique based on the solvent exchange method was implemented using an ultrasonic atomizer system to encapsulate a protein drug in mild conditions. The reservoir-type microcapsules encapsulating lysozyme as a model protein were prepared by inducing collisions between the aqueous droplets containing lysozyme and the droplets of organic solvent with dissolved poly(lactic acid-co-glycolic acid) (PLGA).The main focus of the study was to examine formulation variables on the size and the encapsulation efficiency of the formed microcapsules. The formulation variables examined were concentrations of mannose in the aqueous cores, NaCl in the aqueous collection medium, and PLGA in organic solvent. The mean diameter of the microcapsules ranged from 40 µm to 100 µm. Smaller microcapsules showed lower encapsulation efficiencies. The resulting microcapsules released native lysozyme in a sustained manner, and the release rate was dependent on the formulation conditions, such as the concentration and molecular weight of the polymer used. The solvent exchange method does not induce lysozyme aggregation and loss of its biological activity. The solvent exchange method, implemented by the ultrasonic atomizer system, provides an effective tool to prepare reservoir-type microcapsules for delivering proteins. . † Present address: Department of Advanced Polymer and Fiber Materials, Kyung Hee University, Gyeonggi-do 449-701, South Korea. (1) Bartus, R. T.; Tracy, M. A.; Emerich, D. F.; Zale, S. E. Sustained delivery of proteins for novel therapeutic agents. Science 1998, 281, 1161-1162. (2) Sinha, V. R.; Trehan, A. Biodegradable microspheres for protein delivery. J. Controlled Release 2003, 90, 261-280. (3) Tamber, H.; Johansen, P.; Merkle, H. P.; Gander, B. Formulation aspects of biodegradable polymeric microspheres for antigen delivery.
Journal of Pharmaceutical Investigation, 2012
Spray-dried microspheres based on polysaccharides were developed and the conformational stability and controlled release of incorporated protein were evaluated using bovine serum albumin (BSA) as a model protein. Microspheres composed of water soluble chitosan (WCS), hydroxypropyl-b-cyclodextrin (HP-b-CD) and polyethylene glycol (PEG) were prepared by spray dying. WCS was used as a mucoadhesive and biocompatible polymer. HP-b-CD and PEG were used as protein stabilizer during the spray drying process. Microspheres with 6-7 lm of mean diameter were successfully developed. Encapsulation efficiency of BSA in microsphere was over 70 %. Primary, secondary and tertiary structure of incorporated BSA in microsphere was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence intensity measurement, respectively. Conformational stability of BSA was maintained during the spray drying process. BSA release from microspheres was evaluated in in vitro model using the Transwell Ò insert, and showed a sustained release profile compared to naive BSA. Thus, these microspheres could possibly serve as an optimized delivery system for preserved stability and sustained release of protein.
Journal of Controlled Release, 2005
Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for a-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a h-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.
Journal of Pharmaceutical Sciences, 2007
Novel macromolecular therapeutics such as peptides, proteins, and DNA are advancing rapidly toward the clinic. Because of typically low oral bioavailability, macromolecule delivery requires invasive methods such as frequently repeated injections. Parenteral depots including biodegradable polymer microspheres offer the possibility of reduced dosing frequency but are limited by the inability to adequately control delivery rates. To control release and investigate release mechanisms, we have encapsulated model macromolecules in monodisperse poly(D,L-lactide-co-glycolide) (PLG) microspheres using a double-emulsion method in combination with the precision particle fabrication technique. We encapsulated fluorescein-dextran (F-Dex) and sulforhodamine B-labeled bovine serum albumin (R-BSA) into PLG microspheres of three different sizes: 31, 44, and 80 µm and 34, 47, and 85 µm diameter for F-Dex and R-BSA, respectively. The in vitro release profiles of both compounds showed negligible initial burst. During degradation and release, the microspheres hollowed and swelled at critical time points dependant upon microsphere size. The rate of these events increased with microsphere size resulting in the largest microspheres exhibiting the fastest overall release rate. Monodisperse microspheres may represent a new delivery system for therapeutic proteins and DNA and provide enhanced control of delivery rates using simple injectable depot formulations. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 1176–1191, 2007
Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres
European Journal of Pharmaceutics and Biopharmaceutics, 2008
Proteins were precipitated to ensure their stability upon subsequent encapsulation within PLGA microspheres. Spherical, nanosized protein particles were formed by the addition of a salt (sodium chloride) and a water-miscible organic solvent (glycofurol) to protein solutions. Various process parameters were modified to optimize the precipitation efficiency of four model proteins: lysozyme, a-chymotrypsin, peroxidase and b-galactosidase. As monitored by enzymatic activity measurement of the rehydrated particles, conditions to obtain more than 95% of reversible precipitates were defined for each protein. The study of the structure of the rehydrated particles by absorbance spectroscopy, fluorescence spectroscopy and circular dichroism showed an absence of structural-perturbation after precipitation. Protein particles were then microencapsulated within PLGA microspheres using s/o/w technique. The average encapsulation yield was around 80% and no loss of protein activity occurred after the encapsulation step. Additionally, a lysozyme in vitro release study showed that all of the released lysozyme was biologically active. This method of protein precipitation is appropriate for the encapsulation in PLGA microspheres of various proteins without inactivation.
Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications
Journal of Controlled Release, 2012
Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight -Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C 60 . Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 μm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.
Journal of Biomaterials Science-polymer Edition, 1997
Poly(ε-caprolactone) (PCL) microspheres containing c. 3% bovine serum albumin (BSA) were prepared by melt encapsulation and solvent evaporation techniques. PCL, because of its low Tm, enabled the melt encapsulation of BSA at 75°C thereby avoiding potentially toxic organic solvents such as dichloromethane (DCM). Unlike the solvent evaporation method, melt encapsulation led to 100% incorporation efficiency which is a key factor in the microencapsulation of water-soluble drugs. Examination of the stability of the encapsulated protein by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that protein integrity was unaffected by both methods of encapsulation. In vitro release of the protein into phosphate buffer examined at 37 °C from microspheres prepared by both techniques showed that the release rate from melt-encapsulated microspheres was somewhat slower compared to the release from solvent-evaporated spheres. Both released around 20% of the incorporated protein in 2 weeks amounting to approximately 6.5 µgmg-1 of microspheres. Although the diffusivity of macromolecules in PCL is rather low, it is shown that PCL microspheres are capable of delivering sufficient quantity of proteins by diffusion for prolonged periods to function as a carrier for many vaccines. Unlike poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) polymers which generate extreme acid environments during their degradation, the delayed degradation characteristics of PCL do not generate an acid environment during protein release and, therefore, may be advantageous for sustained delivery of proteins and polypeptides.