High Concentration Large Molecules for Subcutaneous Delivery

Editor’s Take: High-concentration biologics are driving new formulation, manufacturing and device strategies to overcome viscosity, stability and subcutaneous delivery constraints.

Formulation of high dose large molecules like proteins and biologics for injectables has created a great opportunity for unmet medical needs.¹ Drug concentrations at and above 100 mg/mL are considered as high dose, whereas, at or above 250 mg/mL are considered as ultra-high dose with injection volumes of about 2 mL.²

The complexity with high dose formulations stems from aggregation, viscosity, particle formation, long term stability and efficacy of drug products. High dose and smaller volumes of proteins/biologics are often desired for higher clinical efficacy for treatment of life-threatening ailments like rheumatoid arthritis, psoriasis, and neurological disorders, which are preferably administered by subcutaneous (SC). Other factors such as syringeability and injectability are also governed by concentration of drugs in certain injection volumes. Over 46 high concentration antibodies or biologics marketed thus far, about 34 of them are approved by SC, and only 6 by intravenous (IV), and 1 by intramuscular (IM) routes of administration.³

The pain sensation at the injection site is very common. Usach et al. proposed that maximum volume of an SC injection is 1.5 mL with exception of no more than 4 mL if necessary as higher volume is typically associated with the injection site pain.⁴ Berteau et al. evaluated different viscosities in SC to monitor the pain at injection site and observed that highly viscous liquids with viscosity 15–20 cP caused less pain and were easily tolerated as opposed to those having viscosity 1 cP or 8–10 cP.⁵

Fransson et al. investigated the effect of pH and buffer strength and found that at neutral pH 7 and buffer strength as low as possible, the SC formulation was better tolerated upon injection.⁶ Phosphate and citrate buffers with concentration 10 mM and 7.3 mM, respectively, caused less pain at the injection site.⁴ It is, therefore, necessary to maintain pH and buffer strength, and keep low volume for SC high dose proteins or biologics.

Those SC administrations are typically used in 2 mL volume in prefilled syringe (PFS) or autoinjector (AI). Low volume, high dose SC formulation poses some challenges and to overcome those, a few approaches are used including (i) adding viscosity reducing agent to target drug concentrations as low as 100 mg and as high as 250 mg/mL (ii) use novel technologies to deliver the protein concentration as high as 250 mg/mL or higher, and (iii) use a large delivery device with the capability to deliver 2 mL to 40 mL volume.

Formulation development of high concentration biologics

Drug development requiring formulation, analysis and manufacturing can lead to multiple challenges associated with high concentrations including aggregation that negatively impacts the product’s quality, efficacy, stability and safety, and long-term immunogenicity.⁷ Physical characterization tools are necessary to understand the protein-protein interactions. Ultracentrifugation or diafiltration or tangential flow filtration can yield higher concentrations. Increasing concentrations can lead to increased protein-protein interactions, which could impact on conformational and/or colloidal stability and aggregation.

Differential scanning calorimetry (DSC) can be used to quantify free energy difference (ΔG) between natural and denatured proteins and understand the conformational stability; higher ΔG, means greater the stability of proteins or vice versa. Dynamic light and static light scattering can be used to measure particle size and colloidal stability. Higher colloidal stability means the proteins maintain stability as a monomer in native state or partially denatured states due to repulsive or electrostatic interaction.⁸

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Analytical challenges can be overcome by utilizing rapid and simultaneous liquid chromatography and mass spectrometry methods for determination of degradants or any other impurities. An understanding of the mechanisms of protein and excipient degradation and their interactions in solutions is equally important for selecting the appropriate bio-analytical methods to determine formulation stability. Polysorbates, for example, are known to form reactive oxygen species and may react with proteins to cause significant degradation. In cases like these, LC-MS could be used to quantify the chemical degradants.

Physical degradants like conformational changes, denaturation and protein surface adsorption can also be detected by appropriate techniques including DSC and DLS. Other complementary techniques such as differential scanning fluorometry (DSF) can also be used to understand thermal stability, NMR for structural characterization, FTIR for examining the amide I and II bonds of proteins to probe the secondary structure, while circular dichroism (CD) can be used to understand the secondary and tertiary structures of proteins.⁹

Manufacturing challenges often result from lack of appropriate equipment and/or facility. Clear guidance, therefore, is required for scale-up and manufacturing high concentration biologics. For example, vessel design and size with appropriate shaft or mixer can all play an important role for high concentration formulations. Excessive mixing can increase the chances for degradation of biologics, especially at higher concentrations. Thus, the main emphasis is to reduce processing time by maximizing flux rates under controlled temperature and pressure to further curtail the risks for potential degradation in either aqueous or non-aqueous manufacturing media.⁹

Processing techniques such as spray drying, solvent extraction, dehydration, ionic liquids among others are used for manufacturing non-aqueous based suspensions. Spray drying, for example, is applied for creating small spherical fine powder with 5–30 microns in size with the intent to provide better shear thinning and injectability and to protect moisture sensitive drugs as well. The dried powder is then suspended in non-aqueous injectable vehicle preferably in high concentrations (100–250 mg/mL) with desired viscosity (<20 cP or less) and to help improve stability by reducing aggregation and minimizing risks from inherent hydrolytic degradation.

Low temperature solvent extraction and dehydration technique can also be used for thermally labile drugs. This anti-solvent technique results in an amorphous powder by precipitation wherein drug particles are embedded in polymeric matrix, allowing excellent control over particle size, morphology, and surface characteristics with abilities to maintain colloidal stability in non-aqueous suspensions. Shire et al developed a low-shear, highly concentrated biologic suspension by particle engineering of a protein for subcutaneous injection through small gauge needle by reducing the injection force.¹⁰

Manufacturing requirements for ISO cleanrooms

Table 1 lists the cleanrooms with airborne particulate classification requirements for microbial limit in sterile drug products.¹¹

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Marketed high-concentration biologics

Table 2 lists of marketed high concentrate subcutaneous proteins and biologics.

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Novel technologies and ionic liquids for high concentrations biologics

HILOPRO technology is used to achieve the drug concentrations as high as 250 mg/mL by reducing the viscosity of mAbs liquid < 20 cP by adding two ingredients, nicotine and tryptophan in 20 mg or less.¹² Comera Life Sciences, for example, utilized caffeine to reduce the viscosity 37 cP or less to achieve the concentration as high as 200 mg/mL of two mAbs: ipilimumab and infliximab.¹³ Avantor used bis acetyl lysine and propionyl serine to reduce viscosity and increase concentration and also to improve shelf life and stability of proteins.

Xeris Pharmaceutical utilized its Xeriject spray drying and particle engineering technology to achieve crystalline suspensions with concentrations as high as 400 mg/mL, while Lindy Biosciences used its particle engineering microglassification technology in organic solvents to achieve spherical dense, stable particles with drug concentrations as high as 300 mg/mL and 600 mg/mL. Elektrofi, on the other hand, used its Electroject technology to achieve ultra-high drug concentrations; 500 mg/mL and 700 mg/mL microparticle-based stable suspensions by reducing protein-protein interactions and maintaining its injectability.

ENHANZE, a subcutaneous proprietary technology by Halozyme, depolymerizes hyaluronan enzyme to improve mobility of protein solutions, thus allowing much greater volumes (ca.5–10 mL) of high concentration therapeutic doses of mAbs including Herceptin Hylecta (trastuzumab and hyaluronidase), Phesgo (pertuzumab, trastuzumab and hyaluronidase), among others.¹⁴

Ionic liquids (ILs) possess an ability to dissolve challenging poorly soluble molecules, therefore, have been used in formulation and drug delivery in modern times. ILs are considered environmentally green solvents since they are derived from salts and exist as liquids at temperatures below 100ºC. Created by combining cation and anion entities, ILs are stable, non-flammable and act as non-volatile solvents.

Banerjee et al. evaluated CAGE, an IL derived from choline and geranyl acid as salt, in oral insulin formulation and found that it was absorbed through intestinal membrane via modulation of intestinal mucus properties.¹⁵ In another study, Sundaram et al. used ILs derived from choline and amino acids for oral delivery of insulin and found that depending upon concentrations of amino acids, some amino acids have more profound effect on secondary structure of insulin. Other ILs comprised of choline-glycine and choline-alanine formed appreciable amounts of aggregates at high concentrations.¹⁶

Table 3 lists a range of ionic liquids derived from reacting cation and anion moieties.¹⁷

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Aseptic spray drying for high concentration biologics

Aseptic spray drying was first used in 2006 for Exubera by Pfizer for an insulin inhaled powder. Other examples include Verity Pharmaceutical’s triptorelin pamoate (Trelsatar LA) as intramuscular microsphere suspension for prostate cancer, lanreotide acetate (Somatuline LA microspheres by Ispen, Raplixa, a biologic for topical formulation by ProFibrix BV and levodopa (Inbrija) by Acorda Therapeutics.¹⁸

Conclusion

Ascendia offers the expertise in high concentration and highly viscous formulations (solutions and suspensions) of small and large molecules and biologics, and with its state-of-the-art manufacturing facility equipped with fill-finish capabilities of vials, prefilled syringes and cartridges, Ascendia can help expedite the development in the clinical stages of drug products in ISO 5, 6 and 7 (Grade A, B and C) clean rooms. Ascendia’s aseptic process to fill high concentration liquids in vials, prefilled syringes and cartridges can be achieved using peristaltic and piston pumps. Ascendia’s aseptic spray drying can help expedite the formulation development of small and large molecules, proteins and biologics.

References

1. R.G. Strickley and W.J. Lambert, A review of formulations of commercially available antibodies, J. Pharm. Sci., 2021, 110, 2590-2608.

2. I. Ghosh, D. Miranda, T. A. Kulkarni, S. Deodhar, S. Tummala and D. Bindra, Development roadmap for subcutaneous delivery of high dose biologics – high concentration formulation, analytical comparability and patient preference considerations for large volume devices, J. Pharm. Sci., 2025, 114, 103914.

3. I. Ghosh, H. Gutke, M. E. Krause, R. Clemens and R. S. Kashi, A systematic review of commercial high concentration antibody drug products approved in the US: formulation composition, dosage form design and primary packaging considerations, MABS, 2023, 15, 2205540.

4. I. Usach, R. Martinez, T. Festini, and J. S. Peris, Subcutaneous injection of drugs: literature review of factors influencing pain sensation at the injection site, Adv. Ther. 2019, 36, 2986-2996.

5. C. Berteau, O. Filipe-santos, T. Wang, H. E. Rojas, E. Granger, and F. Schwarzenbach, Evaluation of the impact of viscosity, injection volume, and injection flow rate on subcutaneous injection tolerance, Med. Devices (Auckl), 2015, 8, 473-484.

6. J. Fransson and A. Espander-Jansson, Local tolerance of subcutaneous injections, J. Pharm Pharmacol., 1996, 48, 1012-1015.

7. W. Wang and S. Nema, and T. Teagarden, Protein aggregation – mechanism, detection and control, Int. J. Pharm., 2010, 390, 89-99.

8. B. Johnson and A. Rostovtsev, High concentration biologic formulations: Challenges and solutions, Drug Discovery and Development, June 29, 2017.

9. T. A. Khan, D. Bhattacharya, T. R. Christian, M. Holstein, X. Hua, R. James, B. Jiang, A. Josowitz, D. Laiacona, S. Mehta, A. More, E. Mullen, M. Myers, B. Ricart, T. Rickenbacher, Y. Su and N. K. R. Yaragudi, Adv. Drug Deliv. Rev., 2026, 235, 115885.

10. S. J. Shire, Z. Shahrokh and J. Liu, Challenges in the development of high protein concentration formulations, J. Pharm. Sci., 2004, 93, 1390-1402.

11. Who expert committee on specifications for pharmaceutical preparations: Annex 6: good manufacturing practices for sterile products. Who Tech Rep Ser. 2002, # 902:76-93.

12. Bhami’s Research: https://bhamilab.com/index.php/hilopro.

13. Y. Zeng, S. Naik, T. Tran, P. Wuthrich, N. Muni, and R. P. Mahoney, Preclinical pharmacokinetic study on caffeine as an excipient for monoclonal antibody formulations, J. Pharm. Sci., 2023, 112, 2933-2937.

14. I. Ghosh, S. Deodhar, H. Gutke, S. Sridharan, and D. Bindra, Subcutaneous drug delivery of high concentration antibody products – part 2: formulation, device options, and clinical bridging strategies for patient-centric commercial presentations, MABS, 2026, 18, 2680773.

15. A. Banerjee, K. Ibsen, T. Brown, R. Chen, C. Agatemor, and S. Mitragotri, Ionic liquids for oral insulin delivery, Proc. Natl. Acad. Sci., 2018, 115, 7296-7301.

16. V. Sundaram, R. N. Ramanan, M. Selvaraj, R. Vijyayraghavan, D. R. MacFarlane and C. W. Ooi, Enhanced structural stability of insulin aspart in cholinium aminoate ionic liquids, Int. J. Bio. Macromol. 2022, 208, 544-552.

17. M. Guncheva, Role of ionic liquids on stabilization of therapeutic proteins and model proteins, Protein J., 2022, 41, 369-380.

18. S. Ali and J. Huang, Aseptic spray drying for poorly soluble molecules, J. Nanomedicine, 2026, 9, 1076.

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