Thermostabilizing Functional Proteins with Matrix-Assisted Room-Temperature Drying

Yejiong Yu; Siqi Dai; Johnny Xiangyi Zhou; Wei E. Huang; Zhanfeng Cui

doi:10.1016/j.eng.2025.08.045

Engineering ›› :202508045 DOI: 10.1016/j.eng.2025.08.045

Research

research-article

Thermostabilizing Functional Proteins with Matrix-Assisted Room-Temperature Drying

Author information +

History +

PDF (2359KB)

Abstract

This study evaluated the efﬁcacy of the proposed matrix-assisted room-temperature (MART) drying as an alternative to freeze-drying for the thermostabilization of functional proteins in a solid state. To achieve this, protective agents were formulated with functional proteins, and the mixture was dried on a biocompatible cellulose ﬁber matrix. Drying was carried out at room temperature or elevated temperatures (∼ 30 ° C), either through dry air circulation (MART-DA drying) or under a vacuum (MART-V drying). The entire drying process did not involve any refrigeration or freezing steps. The results demonstrated the successful thermostabilization of lactate dehydrogenase (LDH), ﬁbroblast growth factor-2 (FGF-2), and functional enzymes in reverse transcription loop-mediated isothermal ampliﬁcation (RT-LAMP) reagents through the use of MART drying. The functional proteins were immobilized and effectively encapsulated in sugar glass ﬁlms, preserving the proteins’ native structure and functions. The sugar glass ﬁlms were supported by a low-cost three-dimensional (3D) cellulose ﬁber matrix. Overall, MART drying offers a simple, fast, low energy-consumption, and cost-effective strategy for drying functional proteins for long-term room-temperature storage.

Keywords

Protein stabilization / Matrix-assisted room temperature drying / Bioproduct drying / Lactate dehydrogenase / Fibroblast growth factor-2 / RT-LAMP reagents

Cite this article

Download citation ▾

Yejiong Yu, Siqi Dai, Johnny Xiangyi Zhou, Wei E. Huang, Zhanfeng Cui. Thermostabilizing Functional Proteins with Matrix-Assisted Room-Temperature Drying. Engineering 202508045 DOI:10.1016/j.eng.2025.08.045

登录浏览全文

4963

注册一个新账户忘记密码

References

Publishing order | Descend order by publishing year | Descend order by cited within

[1]	Modarres HP, Mofrad MR, Sanati-Nezhad A. Protein thermostability engineering. RSC Adv 2016; 6(116):115252-70.

[2]	Ghosh K, Dill K. Cellular proteomes have broad distributions of protein stability. Biophys J 2010; 99(12):3996-4002.

[3]	Brown LR. Commercial challenges of protein drug delivery. Expert Opin Drug Deliv 2005; 2(1):29-42.

[4]	Sykes C. Time- and temperature-controlled transport: supply chain challenges and solutions. Pharm Ther 2018; 43(3):154-7, 170.

[5]	Frokjaer S, Otzen DE. Protein drug stability: a formulation challenge. Nat Rev Drug Discov 2005; 4(4):298-306.

[6]	Singh SK. Storage considerations as part of the formulation development program for biologics. Am Pharm Rev 2007; 10(3):26-33.

[7]	Webb SD, Webb JN, Hughes TG, Sesin DF, Kincaid AC. Freezing bulk-scale biopharmaceuticals using common techniques and the magnitude of freeze-concentration. BioPharm 2002; 15(5):22-34.

[8]	Singh SK, Kolhe P, Wang W, Nema S. Large-scale freezing of biologics: a practitioner’s review, part 2: practical advice. Bioprocess Int 2009; 7(10):34-42.

[9]	Franks F. Freeze-drying of bioproducts: putting principles into practice. Eur J Pharm Biopharm 1998; 45(3):221-9.

[10]	Liu J, Viverette T, Virgin M, Anderson M, Dalal P. A study of the impact of freezing on the lyophilization of a concentrated formulation with a high ﬁll depth. Pharm Dev Technol 2005; 10(2):261-72.

[11]	Dixon D, Tchessalov S, Barry A, Warne N. The impact of protein concentration on mannitol and sodium chloride crystallinity and polymorphism upon lyophilization. J Pharm Sci 2009; 98(9):3419-29.

[12]	Thorat AA, Suryanarayanan R. Characterization of phosphate buffered saline (PBS) in frozen state and after freeze-drying. Pharm Res 2019; 36(7):98.

[13]	Shalaev E. The impact of buffer on processing and stability of freeze-dried dosage forms. I. Solution freezing behavior. Am Pharm Rev 2005; 8(5):80-7.

[14]	Arsiccio A, Pisano R. The ice-water interface and protein stability: a review. J Pharm Sci 2020; 109(7):2116-30.

[15]	Kolhe P, Holding E, Lary A, Chico S, Singh SK. Large-scale freezing of biologics: understanding protein and solute concentration changes in a cryovessel—part 2. BioPharm Int 2010; 23(7):40-9.

[16]	Thielmann A, Viehmann A, Weltermann BM. Effectiveness of a web-based education program to improve vaccine storage conditions in primary care (keep cool): study protocol for a randomized controlled trial. Trials 2015; 16(1):301.

[17]	Chesko J, Fox C, Dutill T, Vedvick T, Reed S. Lyophilization and stabilization of vaccines. In: Singh M, Srivastava IK, editors. evelopment of vaccines:from discovery to clinical testing. Hoboken: John Wiley & Sons, Inc.; 2011. p. 385-97.

[18]	Kartoglu U, Milstien J. Tools and approaches to ensure quality of vaccines throughout the cold chain. Expert Rev Vaccines 2014; 13(7):843-54.

[19]	Kumru OS, Joshi SB, Smith DE, Middaugh CR, Prusik T, Volkin DB. Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies. Biologicals 2014; 42(5):237-59.

[20]	Larena Fernández I, Vara Callau M, Peña Blasco G, Atance Melendo E, Gay Gasanz B, Blasco Pérez-Aramendía MJ. Vaccine cold chain interruption in a primary care centre and economic evaluation. Enferm Clín Engl Ed 2017; 27(1):44-8.

[21]	Wolfson LJ, Gasse F, Lee-Martin SP, Lydon P, Magan A, Tibouti A, et al. Estimating the costs of achieving the WHO-UNICEF Global Immunization Vision and Strategy, 2006-2015. Bull World Health Organ 2008; 86(1):27-39.

[22]	Cheyne J. Vaccine delivery management. Rev Infect Dis 1989; 11(Suppl 3):S617-22.

[23]	Bishai DM, Bhatt S, Miller LT, Hayden GF. Vaccine storage practices in pediatric ofﬁces. Pediatrics 1992; 89(2):193-6.

[24]

Zhang J, Pritchard E, Hu X, Valentin T, Panilaitis B, Omenetto FG, et al. Stabilization of vaccines and antibiotics in silk and eliminating the cold chain. Proc Natl Acad Sci USA 2012; 109(30):11981-6 [Retraction in: Zhang J, Pritchard E, Hu X, Valentin T, Panilaitis B, Omenetto FG, et al. Proc Natl Acad Sci USA 2016;113(26):E3810].

[25]	Rathore N, Rajan RS. Current perspectives on stability of protein drug products during formulation, ﬁll and ﬁnish operations. Biotechnol Prog 2008; 24(3):504-14.

[26]	Jain NK, Sahni N, Kumru OS, Joshi SB, Volkin DB, Russell MC. Formulation and stabilization of recombinant protein based virus-like particle vaccines. Adv Drug Deliv Rev 2015;93:42-55.

[27]	Balcão VM, Vila MMDC. Structural and functional stabilization of protein entities: state-of-the-art. Adv Drug Deliv Rev 2015;93:25-41.

[28]	Cicerone MT, Pikal MJ, Qian KK. Stabilization of proteins in solid form. Adv Drug Deliv Rev 2015;93:14-24.

[29]	Emami F, Vatanara A, Park EJ, Na DH. Drying technologies for the stability and bioavailability of biopharmaceuticals. Pharmaceutics 2018; 10(3):131.

[30]	Grasmeijer N, Tiraboschi V, Woerdenbag HJ, Frijlink HW, Hinrichs WLJ. Identifying critical process steps to protein stability during spray drying using a vibrating mesh or a two-fluid nozzle. Eur J Pharm Sci 2019;128:152-7.

[31]	Roy I, Gupta MN. Freeze-drying of proteins: some emerging concerns. Biotechnol Appl Biochem 2004; 39(2):165-77.

[32]	Merivaara A, Zini J, Koivunotko E, Valkonen S, Korhonen O, Fernandes FM, et al. Preservation of biomaterials and cells by freeze-drying: change of paradigm. J Control Release 2021;336:480-98.

[33]	Hansen LJJ, Daoussi R, Vervaet C, Remon JP, De Beer TRM. Freeze-drying of live virus vaccines: a review. Vaccine 2015; 33(42):5507-19.

[34]	Luthra S, Obert JP, Kalonia DS, Pikal MJ. Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer. J Pharm Sci 2007; 96(1):61-70.

[35]	Liu J. Physical characterization of pharmaceutical formulations in frozen and freeze-dried solid states: techniques and applications in freeze-drying development. Pharm Dev Technol 2006; 11(1):3-28.

[36]	Telikepalli S, Kumru OS, Kim JH, Joshi SB, O’Berry KB, Blake-Haskins AW, et al. Characterization of the physical stability of a lyophilized IgG1 mAb after accelerated shipping-like stress. J Pharm Sci 2015; 104(2):495-507.

[37]	Stratta L, Capozzi LC, Franzino S, Pisano R. Economic analysis of a freeze-drying cycle. Processes 2020; 8(11):1399.

[38]	Sun D, Cao C, Li B, Chen H, Cao P, Li J, et al. Study on combined heat pump drying with freeze-drying of Antarctic krill and its effects on the lipids. J Food Process Eng 2017; 40(6):e12577.

[39]	Yu Y. Matrix assisted room temperature and vacuum drying of functional proteins and applications [dissertation]. Oxford: University of Oxford; 2021.

[40]	Alcock R, Cottingham MG, Rollier CS, Furze J, De Costa SD, Hanlon M, et al. Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass. Sci Transl Med 2010; 2(19):19ra12.

[41]	Davidson T. Vaccines: history, science, and issues (the story of a drug). Santa Barbara: ABC-Clio/Greenwood; 2017.

[42]	Leung V, Szewczyk A, Chau J, Hosseinidoust Z, Groves L, Hawsawi H, et al. Long-term preservation of bacteriophage antimicrobials using sugar glasses. ACS Biomater Sci Eng 2018; 4(11):3802-8.

[43]	Dulal P, Wright D, Ashﬁeld R, Hill AVS, Charleston B, Warimwe GM. Potency of a thermostabilised chimpanzee adenovirus Rift Valley Fever vaccine in cattle. Vaccine 2016; 34(20):2296-8.

[44]	Leung V, Mapletoft J, Zhang A, Lee A, Vahedi F, Chew M, et al. Thermal stabilization of viral vaccines in low-cost sugar ﬁlms. Sci Rep 2019;9:7631.

[45]	Minamoto K, Nagano M, Inaoka T, Kitano T, Ushijima K, Fukuda Y, et al. Skin problems among ﬁber-glass reinforced plastics factory workers in Japan. Ind Health 2002; 40(1):42-50.

[46]	Dulal P. Investigation of sugar-membrane vaccine stabilisation for improved vaccine thermostability and delivery [dissertation]. Oxford: University of Oxford; 2016.

[47]	Yu Y, Yang A, Ye H, Dye JF, Cui Z. Numerical study of the formation and drying kinetics of a capillary bridge of trehalose solution between two parallel hydrophilic ﬁbres. Chem Eng Sci 2020;226:115849.

[48]	Pikal-Cleland KA, Rodríguez-Hornedo N, Amidon GL, Carpenter JF. Protein denaturation during freezing and thawing in phosphate buffer systems: monomeric and tetrameric b -galactosidase. Arch Biochem Biophys 2000; 384(2):398-406.

[49]	Park H, Park JY, Park KM, Chang PS. Effects of freezing rate on structural changes in L -lactate dehydrogenase during the freezing process. Sci Rep 2021;11:13643.

[50]	Kottakis F, Polytarchou C, Foltopoulou P, Sanidas I, Kampranis SC, Tsichlis PN. FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway. Mol Cell 2011; 43(2):285-98.

[51]	Kotani E, Yamamoto N, Kobayashi I, Uchino K, Muto S, Ijiri H, et al. Cell proliferation by silk gut incorporating FGF-2 protein microcrystals. Sci Rep 2015;5:11051.

[52]	Yu HC, Huang FM, Lee SS, Yu CC, Chang YC. Effects of ﬁbroblast growth factor-2 on cell proliferation of cementoblasts. J Dent Sci 2016; 11(4):463-7.

[53]	Lim B, Ratcliff J, Nawrot DA, Yu Y, Sanghani HR, Hsu CC, et al. Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection. Sci Rep 2021;11:16193.

[54]	Huang WE, Lim B, Hsu CC, Xiong D, Wu W, Yu Y, et al. RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2. J Microbial Biotechnol 2020; 13(4):950-61.

[55]	Bhatnagar BS, Bogner RH, Pikal MJ. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol 2007; 12(5):505-23.

[56]	Cao E, Chen Y, Cui Z, Foster PR. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng 2003; 82(6):684-90.

[57]	Starnes JW. Effect of storage conditions on lactate dehydrogenase released from perfused hearts. Int J Cardiol 2008; 127(1):114-6.

[58]	Carpenter JF, Prestrelski SJ, Arakawa T. Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-speciﬁc stabilization: I. enzyme activity and calorimetric studies. Arch Biochem Biophys 1993; 303(2):456-64.

[59]	Al-Hussein A, Gieseler H. Investigation of histidine stabilizing effects on LDH during freeze-drying. J Pharm Sci 2013; 102(3):813-26.

[60]	Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res 2010; 27(4):544-75.

[61]	Nakanishi K, Sakiyama T, Imamura K. On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J Biosci Bioeng 2001; 91(3):233-44.

[62]	Abdel-Rahman EA, Smejkal Q, Schick R, El-Syiad S, Kurz T. Influence of dextran concentrations and molecular fractions on the rate of sucrose crystallization in pure sucrose solutions. J Food Eng 2008; 84(4):501-8.

[63]	Le Meste M, Champion D, Roudaut G, Blond G, Simatos D. Glass transition and food technology: a critical appraisal. J Food Sci 2002; 67(7):2444-58.

[64]	Liu Y, Bhandari B, Zhou W. Glass transition and enthalpy relaxation of amorphous food saccharides: a review. J Agric Food Chem 2006; 54(16):5701-17.

[65]	Yu L. Amorphous pharmaceutical solids: preparation, characterization and stabilization. Adv Drug Deliv Rev 2001; 48(1):27-42.

[66]	Brawer SA. Theory of relaxation in viscous liquids and glasses. J Chem Phys 1984; 81(2):954-75.

[67]	Surana R, Pyne A, Rani M, Suryanarayanan R. Measurement of enthalpic relaxation by differential scanning calorimetry—effect of experimental conditions. Thermochim Acta 2005; 433(1-2):173-82.

[68]	Putri NI, Van Audenhove J, Kyomugasho C, Van Loey A, Hendrickx M. Relaxation temperature and storage stability of the functionalized cell wall material residue from lemon peel. Food Hydrocoll 2024;150:109711.

[69]	Frauenfelder H, Chen G, Berendzen J, Fenimore PW, Jansson H, McMahon BH, et al. A uniﬁed model of protein dynamics. Proc Natl Acad Sci USA 2009; 106(13):5129-34.

[70]	Kissi EO, Grohganz H, Löbmann K, Ruggiero MT, Zeitler JA, Rades T. Glass-transition temperature of the b -relaxation as the major predictive parameter for recrystallization of neat amorphous drugs. J Phys Chem B 2018; 122(10):2803-8.

[71]	Lai MC, Topp EM. Solid-state chemical stability of proteins and peptides. J Pharm Sci 1999; 88(5):489-500.

[72]	Vallaster B, Engelsing F, Grohganz H. Influence of water and trehalose on a - and b -relaxation of freeze-dried lysozyme formulations. Eur J Pharm Biopharm 2024;194:1-8.

[73]

Starciuc T, Malfait B, Danede F, Paccou L, Guinet Y, Correia NT, et al. Trehalose or sucrose: which of the two should be used for stabilizing proteins in the solid state? A dilemma investigated by in situ micro-Raman and dielectric relaxation spectroscopies during and after freeze-drying. J Pharm Sci 2020; 109(1):496-504.

[74]	Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G. Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze-drying. J Pharm Sci 2010; 99(5):2256-78.

[75]	Schwegman JJ, Hardwick LM, Akers MJ. Practical formulation and process development of freeze-dried products. Pharm Dev Technol 2005; 10(2):151-73.

[76]	Ma GJ, Ferhan AR, Jackman JA, Cho NJ. Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings. Commun Mater 2020;1:45.

[77]	Banerjee I, Pangule RC, Kane RS. Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms. Adv Mater 2011; 23(6):690-718.

[78]	Wang LS, Gupta A, Duncan B, Ramanathan R, Yazdani M, Rotello VM. Biocidal and antifouling chlorinated protein ﬁlms. ACS Biomater Sci Eng 2016; 2(11):1862-6.

[79]

Lin Y, Liu K, Wang C, Li L, Liu Y. Electrochemical immunosensor for detection of epidermal growth factor reaching lower detection limit: toward oxidized glutathione as a more efﬁcient blocking reagent for the antibody functionalized silver nanoparticles and antigen interaction. Anal Chem 2015; 87(16):8047-51.

[80]	Cai B, Hu K, Li C, Jin J, Hu Y. Bovine serum albumin bioconjugated graphene oxide: red blood cell adhesion and hemolysis studied by QCM-D. Appl Surf Sci 2015;356:844-51.

[81]	Steinitz M. Quantitation of the blocking effect of Tween 20 and bovine serum albumin in ELISA microwells. Anal Biochem 2000; 282(2):232-8.

[82]	Besecker J, Cornell KA, Hampikian G. Dynamic passivation with BSA overcomes LTCC mediated inhibition of PCR. Sens Actuators B 2013;176:118-23.

[83]	Wang LS, Gopalakrishnan S, Luther DC, Rotello VM. Protein-based ﬁlms as antifouling and drug-eluting antimicrobial coatings for medical implants. ACS Appl Mater Interfaces 2021; 13(40):48301-7.

[84]	Rubert Pérez CM, Álvarez Z, Chen F, Aytun T, Stupp SI. Mimicking the bioactivity of ﬁbroblast growth factor-2 using supramolecular nanoribbons. ACS Biomater Sci Eng 2017; 3(9):2166-75.

[85]	Cooper ML, Spielvogel RL. Artiﬁcial skin for wound healing. Clin Dermatol 1994; 12(1):183-91.

[86]	Chen WYJ, Rogers AA, Lydon MJ. Characterization of biologic properties of wound fluid collected during early stages of wound healing. J Invest Dermatol 1992; 99(5):550-8.

[87]	Gu TM, Niu XT, Guo H. Clinical observation on changes of ﬁbroblast growth factor in burn wounds. Chin J Repar Reconstr Surg 2000; 14(5):261-3 [Chinese].

[88]	Vogt PM, Lehnhardt M, Wagner D, Jansen V, Krieg M, Steinau HU. Determination of endogenous growth factors in human wound fluid: temporal presence and proﬁles of secretion. Plast Reconstr Surg 1998; 102(1):117-23.

[89]	Ching YH, Sutton TL, Pierpont YN, Robson MC, Payne WG. The use of growth factors and other humoral agents to accelerate and enhance burn wound healing. Eplasty 2011;11:e41.

[90]	Akita S, Akino K, Imaizumi T, Hirano A. Basic ﬁbroblast growth factor accelerates and improves second-degree burn wound healing. Wound Repair Regen 2008; 16(5):635-41.

[91]	Montesano R, Vassalli JD, Baird A, Guillemin R, Orci L. Basic ﬁbroblast growth factor induces angiogenesis in vitro. Proc Natl Acad Sci ISA 1986; 83(19):7297-301.

[92]	Steed DL. The role of growth factors in wound healing. Surg Clin North Am 1997; 77(3):575-86.

[93]	Nissen NN, Polverini PJ, Gamelli RL, DiPietro LA. Basic ﬁbroblast growth factor mediates angiogenic activity in early surgical wounds. Surgery 1996; 119(4):457-65.

[94]	Stewart AA, Byron CR, Pondenis H, Stewart MC. Effect of ﬁbroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. Am J Vet Res 2007; 68(9):941-5.

[95]	Fujimoto Y, Yokozeki T, Yokoyama A, Tabata Y. Basic ﬁbroblast growth factor enhances proliferation and hepatocyte growth factor expression of feline mesenchymal stem cells. Regen Ther 2020;15:10-7.

[96]	Shah DR. Effect of various additives on the stability of basic ﬁbroblast growth factor and development of an intradermal injectable formulation [dissertation]. Kansas City: University of Missouri-Kansas City; 1998.

[97]	Benington L, Rajan G, Locher C, Lim LY. Fibroblast growth factor 2—a review of stabilisation approaches for clinical applications. Pharmaceutics 2020; 12(6):508.

[98]	An K, Liu H, Zhong X, Deng DYB, Zhang J, Liu Z. hTERT-immortalized bone mesenchymal stromal cells expressing rat galanin via a single tetracycline-inducible lentivirus System. Stem Cells Int 2017;2017:6082684.

[99]	Thompson D, Lei Y. Mini review: recent progress in RT-LAMP enabled COVID-19 detection. Sens Actuators Rep 2020; 2(1):100017.

[100]

Yang W, Dang X, Wang Q, Xu M, Zhao Q, Zhou Y, et al. Rapid detection of SARS-CoV-2 using reverse transcription RT-LAMP method. 2020. medRxiv: 2020.03.02.20030130.

[101]

Huang X, Tang G, Ismail N, Wang X. Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva. eBioMedicine 2022;75:103736.

[102]

Yu Y, Zhou JXY, Li B, Ji M, Wang Y, Carnaby E, et al. A quantitative RT-qLAMP for the detection of SARS-CoV-2 and human gene in clinical application. J Microbial Biotechnol 2022; 15(10):2619-30.

[103]

Cui Z, Chang H, Wang H, Lim B, Hsu CC, Yu Y, et al. Development of a rapid test kit for SARS-CoV-2: an example of product design. Biodes Manuf 2020; 3(2):83-6.

[104]

Marino FE, Profﬁtt E, Joseph E, Manoharan A. A rapid, speciﬁc, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in clinical specimens. PLoS One 2022; 17(4):e0266703.

[105]

Dao Thi VL, Herbst K, Boerner K, Meurer M, Kremer LPM, Kirrmaier D, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med 2020; 12(556):eabc7075.