Stability of monoclonal antibodies after simulated subcutaneous administration

Published:March 11, 2021DOI:


      Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.

      Graphical abstract



      AF (artificial fluid), AF+HA (artificial fluid + hyaluronan), BY (brown-yellow), CE-SDS (capillary electrophoresis - sodium dodecyl sulfate), cIEF (capillary isoelectric focusing), CO2 (carbon dioxide), FAL (Float-A-Lyzer), HA (hyaluronan), HMWS (high-molecular weight species), HP-SEC (high performance - size-exclusion chromatography), LMWS (low-molecular weight species), LO (light obscuration), mAb (monoclonal antibody), NTU (nephelometric turbidity units), PBS (phosphate-buffered saline), pI (isoelectric point), RM (reference material), SAL (Slide-A-Lyzer), SbVP (subvisible particle), SC (subcutaneous)
      To read this article in full you will need to make a payment
      APhA Member Login
      APhA Members, full access to the journal is a member benefit. Use your society credentials to access all journal content and features.
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Purchase one-time access:

      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Mathaes R
        • Koulov A
        • Joerg S
        • Mahler HC
        Subcutaneous injection volume of biopharmaceuticals-pushing the boundaries.
        J Pharm Sci. 2016; 105: 2255-2259
        • Sanchez-Felix M
        • Burke M
        • Chen HH
        • Patterson C
        • Mittal S
        Predicting bioavailability of monoclonal antibodies after subcutaneous administration: Open innovation challenge.
        Adv Drug Deliv Rev. 2020;
        • Schuster J
        • Koulov A
        • Mahler HC
        • Detampel P
        • Huwyler J
        • Singh S
        • Mathaes R
        In vivo stability of therapeutic proteins.
        Pharm Res. 2020; 37: 23
        • Richter WF
        • Christianson GJ
        • Frances N
        • Grimm HP
        • Proetzel G
        • Roopenian DC
        Hematopoietic cells as site of first-pass catabolism after subcutaneous dosing and contributors to systemic clearance of a monoclonal antibody in mice.
        MAbs. 2018; 10: 803-813
        • McDonald TA
        • Zepeda ML
        • Tomlinson MJ
        • Bee WH
        • Ivens IA
        Subcutaneous administration of biotherapeutics: Current experience in animal models.
        Curr Opin Mol Ther. 2010; 12: 461-470
        • Rosenberg AS
        Effects of protein aggregates: an immunologic perspective.
        AAPS J. 2006; 8: E501-E507
        • Liu YD
        • Chen Y
        • Tsui G
        • Wei B
        • Yang F
        • Yu C
        • Cornell C
        Predictive In Vitro Vitreous and Serum Models and Methods to Assess Thiol-related Quality Attributes in Protein Therapeutics.
        Anal Chem, 2020
        • Schuster J
        • Mahler HC
        • Koulov A
        • Joerg S
        • Racher AJ
        • Huwyler J
        • Detampel P
        • Mathaes R
        Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS.
        Eur J Pharm Biopharm. 2020; 152: 193-201
        • Schuster J
        • Koulov A
        • Mahler HC
        • Joerg S
        • Huwyler J
        • Schleicher K
        • Detampel P
        • Mathaes R
        Particle analysis of biotherapeutics in human serum using machine learning.
        J Pharm Sci. 2020; 109: 1827-1832
        • Patel S
        • Stracke JO
        • Altenburger U
        • Mahler HC
        • Metzger P
        • Shende P
        • Jere D
        Prediction of intraocular antibody drug stability using ex-vivo ocular model.
        Eur J Pharm Biopharm. 2017; 112: 177-186
        • Awwad S
        • Lockwood A
        • Brocchini S
        • Khaw PT
        The PK-Eye: A novel in vitro ocular flow model for use in preclinical drug development.
        J Pharm Sci. 2015; 104: 3330-3342
        • Bown HK
        • Bonn C
        • Yohe S
        • Yadav DB
        • Patapoff TW
        • Daugherty A
        • Mrsny RJ
        In vitro model for predicting bioavailability of subcutaneously injected monoclonal antibodies.
        J Control Release. 2018; 273: 13-20
        • Kinnunen HM
        • Sharma V
        • Contreras-Rojas LR
        • Yu Y
        • Alleman C
        • Sreedhara A
        • Fischer S
        • Khawli L
        • Yohe ST
        • Bumbaca D
        • Patapoff TW
        • Daugherty AL
        • Mrsny RJ
        A novel in vitro method to model the fate of subcutaneously administered biopharmaceuticals and associated formulation components.
        J Control Release. 2015; 214: 94-102
        • Doell A
        • Schmitz OJ
        • Hollmann M
        Shedding light into the subcutis: A mass spectrometry based immunocapture assay enabling full characterization of therapeutic antibodies after injection in vivo.
        Anal Chem. 2019; 91: 9490-9499
        • Thati S
        • McCallum M
        • Xu Y
        • Zheng M
        • Chen Z
        • Dai J
        • Pan D
        • Dalpathado D
        • Mathias N
        Novel applications of an in vitro injection model system to study bioperformance: Case studies with different drug modalities.
        J Pharm Innov. 2020; 15: 268-280
        • Mach H
        • Gregory SM
        • Mackiewicz A
        • Mittal S
        • Lalloo A
        • Kirchmeier M
        • Shameem M
        Electrostatic interactions of monoclonal antibodies with subcutaneous tissue.
        Ther Deliv. 2011; 2: 727-736
        • Wiig H
        • Swartz MA
        Interstitial fluid and lymph formation and transport: physiological regulation and roles in inflammation and cancer.
        Physiol Rev. 2012; 92: 1005-1060
        • Schaupp L
        • Ellmerer M
        • Brunner GA
        • Wutte A
        • Sendlhofer G
        • Trajanoski Z
        • Skrabal F
        • Pieber TR
        • Wach P
        Direct access to interstitial fluid in adipose tissue in humans by use of open-flow microperfusion.
        Am J Physiol. 1999; 276: E401-E408
        • Webb P
        Temperatures of skin, subcutaneous tissue, muscle and core in resting men in cold, comfortable and hot conditions.
        Eur J Appl Physiol Occup Physiol. 1992; 64: 471-476
        • Zhao L
        • Ji P
        • Li Z
        • Roy P
        • Sahajwalla CG
        The antibody drug absorption following subcutaneous or intramuscular administration and its mathematical description by coupling physiologically based absorption process with the conventional compartment pharmacokinetic model.
        J Clin Pharmacol. 2013; 53: 314-325
        • Ryman JT
        • Meibohm B
        Pharmacokinetics of monoclonal antibodies.
        CPT Pharmacometrics Syst Pharmacol. 2017; 6: 576-588
        • Reed RK
        • Lepsoe S
        • Wiig H
        Interstitial exclusion of albumin in rat dermis and subcutis in over- and dehydration.
        Am J Physiol. 1989; 257: H1819-H1827
        • Kinnunen HM
        • Mrsny RJ
        Improving the outcomes of biopharmaceutical delivery via the subcutaneous route by understanding the chemical, physical and physiological properties of the subcutaneous injection site.
        J Control Release. 2014; 182: 22-32
        • Duems-Noriega O
        • Arino-Blasco S
        Subcutaneous fluid and drug delivery: safe, efficient and inexpensive.
        Rev Clin Gerontol. 2015; 25: 117-146
        • Aukland K
        • Reed RK
        Interstitial-lymphatic mechanisms in the control of extracellular fluid volume.
        Physiol Rev. 1993; 73: 1-78
        • Bretag AH
        Synthetic interstitial fluid for isolated mammalian tissue.
        Life Sci. 1969; 8: 319-329
        • Lobo B
        • Lo S
        • Wang YJ
        • Wong RL.
        Method and Formulation for Reducing Aggregation of a Macromolecule Under Physiological Conditions.
        Google Patents, 2014 (In Genentech I, editor, ed.)
        • Rice P
        • Longden I
        • Bleasby A
        EMBOSS: the European molecular biology open software suite.
        Trends Genet. 2000; 16: 276-277
        • Ilias I
        • Tzanela M
        • Nikitas N
        • Vassiliadi DA
        • Theodorakopoulou M
        • Apollonatou S
        • Tsagarakis S
        • Dimopoulou I
        Evidence of subcutaneous tissue lipolysis enhancement by endogenous cortisol in critically Ill patients without shock.
        In Vivo. 2015; 29: 497-499
        • Maggs DG
        • Jacob R
        • Rife F
        • Lange R
        • Leone P
        • During MJ
        • Tamborlane WV
        • Sherwin RS
        Interstitial fluid concentrations of glycerol, glucose, and amino acids in human quadricep muscle and adipose tissue. Evidence for significant lipolysis in skeletal muscle.
        J Clin Invest. 1995; 96: 370-377
        • Kratz A
        • Ferraro M
        • Sluss PM
        • Lewandrowski KB
        Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Laboratory reference values.
        N Engl J Med. 2004; 351: 1548-1563
        • Wike-Hooley JL
        • Van der Zee J
        • van Rhoon GC
        • Van den Berg AP
        • Reinhold HS
        Human tumour pH changes following hyperthermia and radiation therapy.
        Eur J Cancer Clin Oncol. 1984; 20: 619-623
        • Guyton AC
        • Hall JE.
        Textbook of Medical Physiology.
        11th ed. Elsevier Inc, Philadelphia2006
        • Turitto V
        • Slack SM.
        Blood and Related Fluids.
        (editors)in: Murphy W Black J Hastings G Handbook of Biomaterial Properties. Springer, New York2016: 115-124 (ed.)
        • Schmid I
        • Bonnington L
        • Gerl M
        • Bomans K
        • Thaller AL
        • Wagner K
        • Schlothauer T
        • Falkenstein R
        • Zimmermann B
        • Kopitz J
        • Hasmann M
        • Bauss F
        • Haberger M
        • Reusch D
        • Bulau P
        Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.
        Nat Commun Biol. 2018; 1
        • Yang N
        • Tang Q
        • Hu P
        • Lewis MJ
        Use of in vitro systems to model in vivo degradation of therapeutic monoclonal antibodies.
        Anal Chem. 2018; 90: 7896-7902
        • Yin S
        • Pastuskovas CV
        • Khawli LA
        • Stults JT
        Characterization of therapeutic monoclonal antibodies reveals differences between in vitro and in vivo time-course studies.
        Pharm Res. 2013; 30: 167-178
        • Jiang XG
        • Wang T
        • Kaltenbrunner O
        • Chen K
        • Flynn GC
        • Huang G
        Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system.
        Anal Biochem. 2013; 432: 142-154
        • Del Amo EM
        • Rimpela AK
        • Heikkinen E
        • Kari OK
        • Ramsay E
        • Lajunen T
        • Schmitt M
        • Pelkonen L
        • Bhattacharya M
        • Richardson D
        • Subrizi A
        • Turunen T
        • Reinisalo M
        • Itkonen J
        • Toropainen E
        • Casteleijn M
        • Kidron H
        • Antopolsky M
        • Vellonen KS
        • Ruponen M
        • Urtti A
        Pharmacokinetic aspects of retinal drug delivery.
        Prog Retin Eye Res. 2017; 57: 134-185
        • Liu H
        • Nowak C
        • Patel R
        Modifications of recombinant monoclonal antibodies in vivo.
        Biologicals. 2019; 59: 1-5
        • Linthwaite VL
        • Janus JM
        • Brown AP
        • Wong-Pascua D
        • O'Donoghue AC
        • Porter A
        • Treumann A
        • Hodgson DRW
        • Cann MJ
        The identification of carbon dioxide mediated protein post-translational modifications.
        Nat Commun. 2018; 9: 3092
        • Haaverstad R
        • Romslo I
        • Larsen S
        • HO Myhre
        Protein concentration of subcutaneous interstitial fluid in the human leg. A comparison between the wick technique and the blister suction technique.
        Int J Microcirc Clin Exp. 1996; 16: 111-117
        • Varkhede N
        • Bommana R
        • Schoneich C
        • Forrest ML
        Proteolysis and oxidation of therapeutic proteins after intradermal or subcutaneous administration.
        J Pharm Sci. 2020; 109: 191-205
        • Dillon TM
        • Ricci MS
        • Vezina C
        • Flynn GC
        • Liu YD
        • Rehder DS
        • Plant M
        • Henkle B
        • Li Y
        • Deechongkit S
        • Varnum B
        • Wypych J
        • Balland A
        • Bondarenko PV
        Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.
        J Biol Chem. 2008; 283: 16206-16215
        • Leeman M
        • Choi J
        • Hansson S
        • Storm MU
        • Nilsson L
        Proteins and antibodies in serum, plasma, and whole blood-size characterization using asymmetrical flow field-flow fractionation (AF4).
        Anal Bioanal Chem. 2018; 410: 4867-4873
        • Vlasak J
        • Ionescu R
        Fragmentation of monoclonal antibodies.
        MAbs. 2011; 3: 253-263
        • Goetze AM
        • Liu YD
        • Arroll T
        • Chu L
        • Flynn GC
        Rates and impact of human antibody glycation in vivo.
        Glycobiology. 2012; 22: 221-234
        • Richter WF
        • Jacobsen B
        Subcutaneous absorption of biotherapeutics: knowns and unknowns.
        Drug Metab Dispos. 2014; 42: 1881-1889
        • Wiig H
        • Reed RK
        • Tenstad O
        Interstitial fluid pressure, composition of interstitium, and interstitial exclusion of albumin in hypothyroid rats.
        Am J Physiol Heart Circ Physiol. 2000; 278: H1627-H1639
        • Hotzel I
        • Theil FP
        • Bernstein LJ
        • Prabhu S
        • Deng R
        • Quintana L
        • Lutman J
        • Sibia R
        • Chan P
        • Bumbaca D
        • Fielder P
        • Carter PJ
        • Kelley RF
        A strategy for risk mitigation of antibodies with fast clearance.
        MAbs. 2012; 4: 753-760