Engineering Insulin Cold Chain Resilience to Improve Global Access

TLDR: These copolymers demonstrate promise as simple formulation additives to increase the cold chain resilience of commercial insulin formulations, thereby expanding global access to these critical drugs for treatment of diabetes.

Abstract: There are 150 million people with diabetes worldwide who require insulin replacement therapy and the prevalence of diabetes is rising fastest in middle and low-income countries. Current formulations require costly refrigerated transport and storage to prevent loss of insulin integrity. This study shows the development of simple "drop-in" amphiphilic copolymer excipients to maintain formulation integrity, bioactivity, pharmacokinetics and pharmacodynamics for over 6 months when subjected to severe stressed aging conditions that cause current commercial formulation to fail in under 2 weeks. Further, when these copolymers are added to Humulin R (Eli Lilly) in original commercial packaging they prevent insulin aggregation for up to 4 days at 50 degrees Celsius compared to less than 1 day for Humulin R alone. These copolymers demonstrate promise as simple formulation additives to increase the cold chain resilience of commercial insulin formulations, thereby expanding global access to these critical drugs for treatment of diabetes.

Figures

Figure 5: Stressed aging in commercial packaging. a, Humulin is often sold in standardized 10mL glass vials and packaged in cardboard boxes. Here we tested the stabilizing capacity of AC/DC copolymers in commercial packaging conditions under stressed conditions. 10mL vials of U100 Humulin R were diluted to 95 U/mL with the addition of 50 uL of a MoNi23% stock solution (to a final concentration of 0.01 wt.% copolymer) or water (control). Dilution was necessary to add copolymer to the formulation. These vials were replaced in their original boxes and taped to a shaker plate (150 rpm) in a 37 °C (n=1 per formulation) or 50 °C (n=1 per formulation) incubator. Samples were observed and imaged daily. b,c, Transmittance assays for Humulin or Humulin comprising MoNi23% after aging at (b) 37 °C and (c) 50 °C. Single samples (n=1) were tested for each transmittance curve. d, Blood glucose curves and e, maximum change in blood glucose (Δ glucose) in fasted diabetic rats for samples aged at 50 °C. Data is shown as mean ± s.e. Statistical significance between max Δ glucose was assessed using a REML repeated measures mixed model with rat as a random effect and the age of the formulation as a within-subject fixed effect. A post-hoc Tukey HSD test was used to determine statistical significance between aging timepoints and groups.

Figure 2: Experimental insight into the mechanism of AC/DC excipient stabilization. a, Illustration of proposed stabilization mechanism: (i) In commercial Humulin, monomers at the interface have associative interactions. (ii) Alone, MoNi23% occupies the interface without the presence of insulin. (iii) In combination with Humulin formulations, MoNi23% disrupts insulin-insulin surface interactions, providing a mechanism for inhibiting aggregation. b, Surface tension measurements of Humulin, MoNi23% (0.01 wt.%) formulated with formulation excipients, and Humulin formulated with MoNi23% (0.01 wt.%) (n=2). c, Interfacial rheology measurements of Humulin. Measurements for Humulin formulated with MoNi23% (0.01 wt.%) fell below the resolution of the instrument, indicating that there is no protein aggregation at the interface (n=3).

Figure 4: Insulin activity after aging in diabetic rats. Fasted diabetic male rats received subcutaneous administration (1.5U/kg) of each insulin formulation a, Humulin b, Humulin with MoPhe6%, c, Humulin with MpPhe8%, or d, Humulin with MoNi23% at each aging timepoint (0, 2, 4, 6 months). e, Comparison of each formulation a t=0 months. In these assays, 32 rats were randomly assigned to one of the four formulation groups (n=8) and each rat received one dose of the formulation at each aging timepoint in a random order. Blood glucose levels were measured every 30 minutes using a handheld glucose monitor and the change in blood glucose relative to baseline glucose measurements were plotted. Baseline glucose measurements ranged from 300-600 mg/dL (See Supplementary Figure 5 for raw glucose curves). The maximum difference in glucose from baseline (Δ glucose) was also plotted for each formulation as a measure of formulation potency. f, Pharmacokinetics of Humulin and Humulin with MoNi23% at t=0 and t=6 months. All data is shown as mean ± s.e. Statistical significance between max Δ glucose was assessed using a REML repeated measures mixed model with rat as a random effect and the age of the formulation as a within-subject fixed effect. A post-hoc Tukey HSD test was used on Humulin formulations to determine statistical significance between aging timepoints.

Figure 1. Scheme of the cold chain and insulin aggregation mechanism. a, To maintain integrity, commercial insulin formulations must currently be transported and stored in refrigerated containers for the weeks-long duration of worldwide distribution. b, Aggregation mechanism of commercial insulin formulations. The insulin hexamer is at equilibrium with monomers in formulation. These monomers interact at the interface, where the exposure of hydrophobic domains during insulin-insulin interaction nucleate amyloid fiber formation. c, Chemical structure of AC/DC excipients, poly(acryloylmorpholine77%co-N-isopropylacrylamide23%) (MoNi23%). AC/DC excipients have a molecular weight between 2-5 kDa (See Table S1). d, AC/DC excipients are amphiphilic copolymers that adsorb to interfaces, reducing insulininsulin interactions and delaying the nucleation of insulin amyloidosis.

Figure 3: Formulation with AC/DC copolymers stabilizes insulin. a, 1mL of Commercial Humulin or Humulin with the addition of AC/DC excipients (i) MoPhe6%, (ii) MpPhe8%, (iii) MoNi23% were aliquoted into 2mL glass vials and aged at 37°C with constant agitation (150 rpm) for 0, 2, 4, and 6 months. Additional 2 week and 1 month timepoints were added for the Humulin control. All formulations were at a concentration of 95U/mL (diluted so that copolymers could be added to commercial Humulin). b, Transmittance assay to assess the aggregation of proteins in formulation over time by monitoring changes in transmittance at 540nm (n=1 per formulation timepoint). c, In vitro activity by assaying for phosphorylation of Ser473 on AKT after stimulation with either Humulin or MoNi23% at 0 month and 6 month timepoints. Insulin concentrations are shown as Log(ng/mL). d, Log(EC50) values for each formulation. Statistical significance was assessed using the Extra sum-of-squares F-test to determine if Log(EC50) differed between datasets. Data sets were compared in pairs, and Bonferroni post-hoc tests were used to adjust for multiple comparisons (alpha=0.008). e-h, Circular dichroism spectra from 200-260nm for each formulation (diluted to 0.2 mg/mL in PBS) at each time point. c, Results shown are mean ± s.e plotted as a ratio of [pAKT]/[AKT] for each sample (n=3 cellular replicates) and an EC50 regression (log(agonist) vs. response (three parameters)) was plotted using GraphPad Prism 8. d, Statistical significance was assessed using the Extra sum-of-squares F-test to determine if Log(EC50) differed between datasets. Data sets were compared in pairs, and Bonferroni post-hoc tests were used to adjust for multiple comparisons (alpha=0.008).

Full text

1
Engineering Insulin Cold Chain Resilience to
Improve Global Access
Caitlin L. Maikawa
1‡
, Joseph L. Mann
2,‡
, Aadithya Kannan
3
, Catherine M. Meis
2
, Abigail K.
Grosskopf
3
, Ben S. Ou
1
, Anton A. A. Smith
2,4,†
, Gerald G. Fuller
3
, David M. Maahs
6,7
, Eric A.
Appel
1,2,5,7,8
*
‡
These authors contributed equally
1
Department of Bioengineering, Stanford University, Stanford CA 94305, USA
2
Department of
Materials Science & Engineering, Stanford University, Stanford CA 94305, USA
3
Department of Chemical Engineering, Stanford University, Stanford CA 94305, USA
4
Department of Science and Technology, Aarhus University, 8000 Aarhus, Denmark
6
Department of Pediatrics (Endocrinology), Stanford University, Stanford CA 94305, USA
7
Diabetes Research Center, Stanford University, Stanford CA 94305, USA
8
Stanford CHEM-H Institute, Stanford University, Stanford CA 94305, USA
†
Present Address: Department of Health Technology, Technical University of Denmark, 2800
Kgs. Lyngby, Denmark
*
Person to whom correspondence should be addressed:
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 7, 2021. ; https://doi.org/10.1101/2021.04.13.439582doi: bioRxiv preprint

2
Dr. Eric A. Appel, eappel@stanford.edu
KEYWORDS Diabetes, Insulin, Global Access, Drug Development
ABSTRACT There are 150 million people with diabetes worldwide who require insulin
replacement therapy and the prevalence of diabetes is rising fastest in middle and low-income
countries. Current formulations require costly refrigerated transport and storage to prevent loss of
insulin integrity. This study shows the development of simple “drop-in” amphiphilic copolymer
excipients to maintain formulation integrity, bioactivity, pharmacokinetics and pharmacodynamics
for over 6 months when subjected to severe stressed aging conditions that cause current
commercial formulation to fail in under 2 weeks. Further, when these copolymers are added to
Humulin R (Eli Lilly) in original commercial packaging they prevent insulin aggregation for up to
4 days at 50 °C compared to less than 1 day for Humulin R alone. These copolymers demonstrate
promise as simple formulation additives to increase the cold chain resilience of commercial insulin
formulations, thereby expanding global access to these critical drugs for treatment of diabetes.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 7, 2021. ; https://doi.org/10.1101/2021.04.13.439582doi: bioRxiv preprint

3
INTRODUCTION
There are over 150 million people with diabetes requiring insulin replacement therapy
worldwide.
1
The worldwide population of people with insulin-deficient diabetes is increasing at a
rate of 3-5% annually, with the highest increases in prevalence occurring in warm regions such as
Africa, the Western Pacific, and the Middle East.
1-2
Unfortunately, insulin is prone to irreversible
aggregation when exposed to high temperatures and/or agitation and requires careful storage and
refrigerated transport (the cold chain) to retain activity over its shelf life. Maintaining insulin
integrity presents a challenge for the pharmaceutical industry, health care providers, and people
with diabetes worldwide.
3-4
Indeed, annual global costs for the refrigerated transport of all
biopharmaceuticals globally exceed $15 billion (USD), and losses due to interruptions in the cold
chain reach $35 billion (USD) annually.
4
A primary driver of the loss of formulation integrity is the propensity of proteins to
aggregate at hydrophobic interfaces when exposed to elevated temperatures.
5
In the case of insulin
formulations, agitation and elevated temperatures – conditions common to worldwide transport
and cold chain interruptions – increase interactions between partially unfolded insulin monomers
adsorbed to interfaces, leading to nucleation of insulin amyloid fibrils.
6-9
Recent studies in insulin stabilization have relied on covalent or non-covalent attachment
of hydrophilic polymers directly to insulin or through encapsulation of insulin or insulin crystals
in hydrogels.
10-15
While these strategies have successfully shielded insulin from interfacial
adsorption or insulin-insulin interactions and increased insulin stability, they can also lead to
increased absorption times and longer circulation times in vivo. A more translatable stabilization
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 7, 2021. ; https://doi.org/10.1101/2021.04.13.439582doi: bioRxiv preprint

4
method would utilize an inactive excipient that could be incorporated into existing formulations
without altering the drug pharmacokinetics.
Amphiphilic copolymers present an alternative to polymer-protein conjugation, exploiting
their propensity to gather at the air-water interface to hinder insulin-interface interactions.
6, 16-18
Poloxamers have been successfully used to improve insulin stability and have been employed in
commercial formulations (Insuman U400, Sanofi-Aventis). Yet, poloxamers have not been widely
adopted and have not succeeded in reducing reliance on the cold chain for insulin transport.
Previously we have shown that biocompatible and non-toxic acrylamide carrier/dopant
copolymers (AC/DC), a class of amphiphilic copolymers comprising water-soluble “carrier” and
hydrophobic “dopant” monomers, can enable the development of ultra-fast insulin formulations.
19
We hypothesized that these excipients could be applied more broadly to improve the stability of
current commercial insulin formulations in the context of reducing cold chain reliance and
improving formulation resilience. In this work, we aim to understand the stabilization mechanism
of AC/DC copolymer excipients and to test the limits of their stabilizing capacity long-term and
under extreme environmental conditions. We report the ability of select copolymers to act as
simple “drop-in” excipients to stabilize commercial insulin formulations (Humulin R, Eli Lilly)
without altering their bioactivity, pharmacokinetics or pharmacodynamics, constituting an
important step toward improving global access to these critical drugs.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 7, 2021. ; https://doi.org/10.1101/2021.04.13.439582doi: bioRxiv preprint

5
EXPERIMENTAL METHODS
Materials
Humulin R (Eli Lilly) was purchased and used as received. Solvents N,N-dimethylformamide
(DMF; HPLC Grade, Alfa Aeser , >99.7%), hexanes (Fisher, Certified ACS, >99.9%), ether
(Sigma, Certified ACS, Anhydrous,>99%) and CDCl
3
(Acros, >99.8%) were used as received.
Monomers N-(3-methoxypropyl)acrylamide (MPAM; Sigma, 95%), 4-acryloylmorpholine
(MORPH; Sigma, >97%) were filtered with basic alumina prior to use. Monomers N-
phenylacrylamide (PHE; Sigma, 99%) and N-isopropylacrylamide (NIPAM; Sigma, >99%) were
used as received. RAFT chain transfer agents 2-cyano-2-propyl dodecyl trithiocarbonate (2-
CPDT; Strem Chemicals, >97%) and 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-
cyanopentanoic acid (BM1433; Boron Molecular, >95%) were used as received. Initiator 2,2’-
azobis(2-methyl-propionitrile) (AIBN; Sigma, >98%) was recrystallized from methanol (MeOH;
Fisher, HPLC Grade, >99.9%) and dried under vacuum before use. Z-group removing agents
lauroyl peroxide (LPO; Sigma, 97%) and hydrogen peroxide (H2O2; Sigma, 30%) were used as
received. Streptozotocin (99.58%) was purchased from MedChem Express. All other reagents
were purchased from Sigma-Aldrich unless otherwise specified.
Surface Tension
Time resolved surface tension of the air-solution interface was measured with a Platinum/Iridium
Wilhelmy plate connected to an electrobalance (KSV Nima, Finland). The Wilhelmy plate was
partially immersed in the aqueous solution in a Petri dish, and the surface tension of the interface
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 7, 2021. ; https://doi.org/10.1101/2021.04.13.439582doi: bioRxiv preprint

Frequently asked questions

Q1. What have the authors contributed in "Engineering insulin cold chain resilience to improve global access" ?

This study shows the development of simple “ drop-in ” amphiphilic copolymer excipients to maintain formulation integrity, bioactivity, pharmacokinetics and pharmacodynamics for over 6 months when subjected to severe stressed aging conditions that cause current commercial formulation to fail in under 2 weeks. Was not certified by peer review ) is the author/funder. Further, when these copolymers are added to Humulin R ( Eli Lilly ) in original commercial packaging they prevent insulin aggregation for up to 4 days at 50 °C compared to less than 1 day for Humulin R alone. 

Q2. What have the authors stated for future works in "Engineering insulin cold chain resilience to improve global access" ?

22, 30 Future studies will require continued evaluation of the limits of AC/DC stabilized insulin and explore the application of these excipients to other protein therapeutics. 

Q3. How has stress aging been used to test insulin stability?

Stressed aging, through incubationat elevated temperatures with continuous agitation, has been previously used to test insulin stability. 

Q4. What is the primary driver of the loss of insulin integrity?

4A primary driver of the loss of formulation integrity is the propensity of proteins toaggregate at hydrophobic interfaces when exposed to elevated temperatures. 

Q5. What is the effect of interruptions in the cold chain on insulin bioactivity?

While commercial insulin formulations have good shelf lives when stored properly, interruptions in the cold chain can decrease insulin bioactivity and formulation integrity. 

Q6. How long did the preparation of the insulin be aged?

Formulations of Humulin alone or Humulin with an AC/DC excipient added were prepared and aged for 0, 2, 4, or 6 months at 37 °C with constant agitation (150 rpm on an orbital shaker plate). 

Q7. What is the likely mechanism for stability?

No thermo-responsive behavior was observed by the MoNi23% excipient at the temperatures tested in this study (Figure S8), thus disruption of surface interactions remains the most likely mechanism for stability. 

Q8. What is the effect of the active formulations on glucose levels?

Active formulations resulted in a distinct initial drop in blood glucose from extreme hyperglycemia that reached a minimum in the range of normoglycemia between 60-100 minutes after administration (Figure 4, Supplementary Figure 3). 

Q9. What was the way to measure the cell viability of the rats?

Fit parameter F was constrained to 50, Bottom was constrained to 4 (the negative control for the assay) and the Top was constrained to be the same for all data sets (cell viability should be equal for all data sets as polymer concentration approaches 0). 

Q10. What is the effect of the addition of acryloylmorpholine to Humul?

the addition of completely hydrophilic poly(acryloylmorpholine) (Mo) to Humulin did not lower the surface tension, indicating that the amphiphilic copolymer is required to displace insulin (Supplementary Figure 1B). 

Q11. What temperature was used to agitate the cardboard packaging?

The cardboard packaging was affixed to a rotary shaker inside a temperature-controlled incubator and agitated at 150 RPM (Supplementary Figure 7b). 

Q12. What is the effect of MoNi23% on the surface tension of Humulin R?

The decrease in surface tension upon addition of MoNi23% to Humulin indicates that there are more species at the interface when MoNi23% and Humulin are formulated together, compared to Humulin alone. 

Q13. What was the LC50 value of the new vials of Humulin R?

MoNi23 (0.01 wt.%) was added to new vials of Humulin R using a syringe (dilution from 100 U/mL to 95U/mL to allow addition of copolymer; control vial was diluted with water) and the vials were then replaced in the original cardboard packaging with the package insert (Supplementary Figure 7a).