Thermal stability and storage of human insulin

Objectives

This is a protocol for a Cochrane Review (prototype). The objectives are as follows:

To analyse the effects of storing human insulin above or below the manufacturers' recommended insulin temperature storage range or advised usage time, or both, after dispensing human insulin to people with diabetes.

Background

There are two major forms of diabetes, type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM results from an autoimmune process destroying the insulin‐producing beta cells (ß‐cells) of the pancreas. T1DM is the major cause of diabetes in childhood and adolescence, and injections of insulin are necessary for survival. In the tenth revision of its diabetes atlas, the International Diabetes Federation (IDF) estimated that the number of people with diabetes worldwide would increase from 573 million people in 2021 to 783 million people in 2045, representing a 46% increase (IDF 2021). This rise is calculated to be higher in low‐ and middle‐income countries, with projected increases of 134% in Africa (87% in the Middle East and North Africa), 68% in South‐East Asia and 50% in South and Central America (IDF 2021). In comparison, the estimates for Europe, the Western Pacific and North America and the Caribbean are 13%, 27% and 24%, respectively (IDF 2021). 

The peptide hormone insulin was discovered in 1921 by Banting and Best; it was first used in the treatment of diabetes in 1922 and has been included in the World Health Organization's (WHO's) Model List of Essential Medicines since 1977 (WHO EML 2021). T2DM accounts for approximately 90% of diabetes and is associated with insulin resistance: as a consequence of ineffective insulin action to transport glucose into cells, an increased insulin demand develops and over time inadequate insulin production occurs due to exhaustion of pancreatic ß‐cells. T2DM being related to obesity and lack of exercise is initially treated with exercise/diet and oral glucose‐lowering drugs, but insulin may be needed to further improve diabetes management and in case pancreatic ß‐cells are not able to compensate for the increased insulin demand. The need for insulin to treat T2DM is expected to increase by more than 20% from 2018 to 2030 (Basu 2019). Diabetes may also occur during pregnancy (gestational diabetes) or may develop because of diseases of the pancreas, endocrine diseases, infections, drugs, immune disorders or genetic syndromes.

Even 100 years after its discovery, access to insulin still remains a challenge for many populations around the globe. Barriers to access mainly relate to affordability and availability, with costs, also related to associated necessary medical devices (e.g. test‐stripes, syringes, pens), placing a large burden on patients and healthcare systems (Beran 2006; Beran 2016). For people with T1DM, the goal of insulin therapy is to provide insulin that mimics physiological insulin secretion in order to achieve near‐normal glycaemic levels. Insulin is most commonly administered by subcutaneous injection. Insulin is usually applied through insulin syringes, insulin pens or insulin pumps. Since the early 1920s, people with diabetes were treated with insulin, which was purified from bovine or porcine pancreas (animal insulin). Later, recombinant human insulin utilising recombinant deoxyribonucleic acid (DNA) technology (Chance 1993) and insulin analogues (insulin‐like molecules, engineered on the basis of the molecular structure of human insulin by changing amino acid sequences and physiochemical properties) were marketed (Fullerton 2018; Hemmingsen 2021; Semlitsch 2020).

Early data indicated loss of insulin potency depending on storage temperatures (Brange 1987). The stability of human insulin depends upon a number of environmental factors (e.g. purity, pH, humidity, changes in the primary structure of the insulin molecule, added substances for enhancing or prolonging insulin absorption) and is thought to be especially susceptible to temperature and sunlight exposure (Pingel 1972; Storvick 1968; Vimalavathini 2009). These factors may become more important because of increasingly hot weather and heat extremes caused by the worldwide climate crisis (Atwoli 2021; Ebi 2021; Quijal‐Zamorano 2021). Temperature not only affects diabetes control (Westphal 2010) but also the pharmaceutical quality of various drugs, especially of protein and peptide‐based compounds like insulin or monoclonal antibodies used to treat various inflammatory diseases. The potency of insulin is measured in units per millilitre (U/mL), indicating the blood glucose‐lowering activity of an insulin formulation per volume. High‐performance liquid chromatography (HPLC) is the gold standard for insulin concentration and insulin stability assessment (Farid 1989; Fisher 1986). HPLC quantification of the chemical potency of insulin is correlated with its biological activity. Traditionally, in vivo rabbit biopotency assay was used to monitor blood glucose over time (Farid 1989; Fisher 1986; Smith 1985). However, the measured and actual biological potency of insulin might differ. Insulin potency may be impacted by high or low ambient temperature, sunlight and usage beyond the expiration date, as well as exceeding the recommended timeline once opened or in use. For insulin pumps worn close to the body, the temperature in the reservoir is increased, and constant movements may increase fibril formation (Herr 2014; Pryce 2009).

In order to prevent self‐aggregation during transport and storage, zinc ions and phenolic conservative agents are added to insulin formulations to stabilise insulin molecules as hexameric complexes (Brange 1997; Huus 2005; Huus 2006). Agitation or heating may cause the hexamers to dissociate and release monomers which are susceptible to form fibrils (Ahmad 2003; Kurouski 2012). Formation of these insulin polymers is reported to occur up to 10 times faster at 37 °C compared to 25 °C (Weiss 2013). Amyloid fibrils may also exhibit immunogenic capabilities (Brange 1997; Mori 2021). Insulin fibrillation by means of aggregation of insulin molecules also adversely impacts potency (Groenning 2009). If insulin within fibrils is subcutaneously injected, bioavailability is reduced because stable bonds of insulin molecules within fibrils provide less insulin monomers available for resorption (Derewenda 1989). Fibrils may be visible in vials by the naked eye but are difficult to detect in pens or pump reservoirs. If temperature rises insulin's degradation increases, and combination of agitation and heat accelerates formation of agglomerates. Therefore, the advice is to discard insulin formations which appear visually modified (Shnek 1998). However, thermal denaturation is a complex process, correlated with both time and temperature; and it may be partially reversible (Huus 2005). Application of high temperatures for a prolonged time usually leads to denaturation due to irreversible conformational changes of insulin and formation of insulin fibrils (Vimalavathini 2009). In contrast, fluctuating temperatures (compared to continuous heating conditions) may not cause irreversible aggregation of insulin molecules.

In their excellent review, Heinemann and colleagues highlight the complex process of managing insulin transport and storage temperature where manufacturers, distributors, healthcare professionals and health authorities, and finally the patient, all play a part in securing insulin stability, concluding “From our point of view, there is also a need for systematic evaluation of insulin stability at different levels of the cold chain, with a clear focus on pharmacies and storage in patients’ homes” (Heinemann 2020). 

Recommendations by health authorities underline the fact that insulin is temperature sensitive and should be protected from heat and freezing conditions. The American Diabetes Association (ADA) publishes the following advice on its website (ADA 2021):  
"Although manufacturers recommend storing your insulin in the refrigerator, injecting cold insulin can sometimes make the injection more painful. To avoid this, many providers suggest storing the bottle of insulin you are using at room temperature. Insulin kept at room temperature will last approximately one month. Remember though, if you buy more than one bottle at a time to save money, store the extra bottles in the refrigerator. Then, take out the bottle ahead of time so it is ready for your next injection. Here are some other tips for storing insulin:

• Do not store your insulin near extreme heat or extreme cold.

• Never store insulin in the freezer, direct sunlight, or in the glove compartment of a car.

• Check the expiration date before using, and don't use any insulin beyond its expiration date.

• Examine the bottle closely to make sure the insulin looks normal before you draw the insulin into the syringe."

Manufacturers' specifications recommend to keep insulin away from sunlight, not to freeze it, and store it in a refrigerator or at 'room temperature', usually not exceeding 25 °C or 30 °C. Nevertheless, room temperature may be defined differently in various parts of the world. Intact vials of insulin should be stored at low temperatures, i.e. between 2 °C and 8 °C, requiring affordable and reliable refrigeration. However, refrigeration may not be available or possible in various parts of the world (Ogle 2016). Moreover, unlike pharmaceutical refrigerators, household refrigeration may be unreliable and temperatures can drop below the freezing point (Heinemann 2020). Once opened, a vial or cartridge can be stored at ambient temperature and used for approximately four to six weeks. The contents of the vial or cartridge are technically no longer sterile, and it is recommended to use the insulin in as short a time as possible to minimise concerns about potential microbiological contamination once the container has been opened or punctured. However, recommendations differ with regard to advised usage time once open and maximum temperature in‐use on the one side; and type of insulin, brand, insulin concentrations and carrier (vial, pen, cartridge, pump) on the other side (table 1 in Heinemann 2020 and table S1 in Kaufmann 2021). For human insulin, the current shelf life in use may range from 10 days to 45 days and the maximum temperature in‐use recommendations vary between 25 °C and 37 °C. Tropical regions will often exceed these temperature thresholds and due to climate crisis periods of extreme heat will happen more often in almost any part of the world. Whereas WHO (WHO 2011) and regulatory bodies provide guidance how to handle the professional insulin cold chain, there does not seem to be a consensus on the issue of temperature and storage conditions of insulin on patients' level, leading to the possibility that unused insulin may be unnecessarily discarded if people with diabetes strictly follow manufacturers' instructions (Flood 2015; Grajower 2003; Grajower 2014).

For this review, optimal cold‐chain management from manufacturing until the point of delivery to people with diabetes, and adequate storage conditions at the professional healthcare level, are seen as a prerequisite to investigate in detail whether it is possible for consumers to use insulin outside manufacturers' recommended temperature range and especially without refrigeration during the period of use in hot climate conditions. This is particularly important in low‐resource settings to remove "at least one significant barrier" (Kaufmann 2021) aside from the major health equity issues of affordability and availability.

Objectives

To analyse the effects of storing human insulin above or below the manufacturers' recommended insulin temperature storage range or advised usage time, or both, after dispensing human insulin to people with diabetes.

Methods

Inclusion and exclusion criteria

With regard to the objectives we established the following 'Population, Intervention, Comparator, Outcome, Timing' (PICOT) table, as follows.

Item	Definition
Population	Clinical studies: people with type 1 or type 2 diabetes mellitus treated with human insulin
Intervention	Storage of human insulin above or below manufacturers' recommended temperature storage range or advised usage time, or both
Comparator	Storage of human insulin according to manufacturers' recommended temperature storage range or advised usage time, or both
Outcome	Potency and bioactivity parameters (e.g. measured by HPLC, bioassay, assessment of insulin monomer structure, insulin efficiency, structural physical damage, physical damage visible to the naked eye, antimicrobial activity) Clinical parameters (e.g. HbA1c, blood sugar, hypoglycaemic episodes, diabetic ketoacidosis, adverse events including potential additive effects of temperatures and recommended storage periods, health‐related quality of life)
Timing	Temperature studies: any time of exposure Usage time studies: above manufacturers' recommended usage time for the analysed human insulin formulation
HbA1c: glycosylated haemoglobin A1c; HPLC: high performance liquid chromatography

Types of studies

Any clinicall study fulfilling the above‐mentioned PICOT criteria will be included. For this review we are especially interested in studies emulating patients' living conditions, mimicking daily use of human insulin. We will also include laboratory studies investigating storage of human insulin above or below manufacturers' recommended temperature storage range or advised usage time, or both.

Diagnostic criteria for diabetes mellitus

In order to be consistent with changes in the classification of, and diagnostic criteria for, diabetes mellitus over the years, the diagnosis should be established using the standard criteria valid at the time of the study commencing (for example ADA 2003; ADA 2017; WHO 1999). Ideally, the diagnostic criteria should have been described. We will use the study authors' definition of diabetes mellitus if necessary.

Specific exclusion criteria

The following studies will be excluded:

• studies investigating animal insulin;

• studies investigating insulin analogues;

• studies investigating the immediate effects of ambient temperature in people with diabetes (i.e. no focus on storage conditions of human insulin);

• in‐vitro studies (e.g. studies on cells or receptors);

• case reports or case series.

Search methods for identification of studies

Electronic searches

One review author (MIM) developed the search strategies using analytical text‐mining of 24 relevant publications already known to review author BR. We used the tools PubReMiner (hgserver2.amc.nl/cgi-bin/miner/miner2.cgi) and Yale MeSH Analyzer (mesh.med.yale.edu).

We will search the following sources from the inception of each database to the date of search and will place no restrictions on the language of publication:

• Cochrane Central Register of Controlled Trials (CENTRAL) via the Cochrane Register of Studies Online (CRSO);

• MEDLINE (Ovid MEDLINE ALL 1946 to Daily Update);

• CINAHL EBSCO (Cumulative Index to Nursing and Allied Health Literature);

• Science Citation Index Expanded (Web of Science; 1945 to present);

• WHO Global Index Medicus (www.globalindexmedicus.net);

• ClinicalTrials.gov (www.clinicaltrials.gov);

• World Health Organization International Clinical Trials Registry Platform (ICTRP) (www.who.int/trialsearch);

For detailed search strategies, see Appendix 1. We will continuously apply an email alert service for MEDLINE via OvidSP to identify newly published studies using the search strategy detailed in Appendix 1.

Searching other resources

We will contact the three leading insulin‐producing pharmaceutical companies (Eli Lilly, Novo Nordisk, Sanofi) to obtain published and unpublished data on insulin thermostability. We will attempt to identify other potentially eligible studies or ancillary publications by searching the reference lists of included studies, systematic reviews, meta‐analyses, and health technology assessment reports. We will also contact the authors of included studies to obtain additional information on the studies and establish whether we may have missed further studies.

Data collection and analysis

Selection of studies

Two review authors (BR, BB) will independently screen the abstract, title, or both, of every record retrieved by the literature searches. We will obtain the full text of all potentially relevant records. We will resolve disagreements through consensus or by recourse to a third review author (MIM). If we cannot resolve a disagreement, we will categorise the study as 'awaiting classification' and will contact the study authors for clarification. We will present an adapted PRISMA flow diagram to show the process of study selection (Liberati 2009). We will list all articles excluded after full‐text assessment in a 'Characteristics of excluded studies' table and will provide the reasons for exclusion.

Data extraction and management

For studies that fulfil our inclusion criteria, one review author (BR) will extract type of experimental study, study characteristics, stated aim of the study, type and brand of human insulin, use of concomitant oral antidiabetic agents, data on cold chain management, temperature (constant, oscillating or both), light exposure and usage time, container use (unopened, open, in‐use), container type (vial, cartridge, prefilled pens), sterility, injection device (syringe, pen, pump), storage conditions, potency/bioactivity parameters and clinical outcomes.

Another author (BB) will check these data extractions, and we will resolve any disagreements by discussion or, if required, by consultation with a third review author (MIM). We will establish our own context‐specific data extraction sheets after piloting data extraction for five studies. All extracted data and risk of bias evaluations (see below) will be stored online in an open repository (Zenodo.org).

We will provide information, including the study identifier for potentially relevant ongoing trials, in the 'Characteristics of ongoing trials' table. We will email all authors of included studies to enquire whether they would be willing to answer questions regarding their studies. We will present the results of this survey in an appendix. We will thereafter seek relevant missing information on the study from the primary study author(s), if required.

Dealing with duplicate and companion publications

In the event of duplicate publications, companion documents, or multiple reports of a primary study, we will maximise the information yield by collating all available data, and we will use the most complete data set aggregated across all known publications. We will list duplicate publications, companion documents, multiple reports of a primary study, and trial documents of included trials (such as trial registry information) as secondary references under the study identifier of the included study. Furthermore, we will list duplicate publications, companion documents, multiple reports of a study, and trial documents of excluded trials (such as trial registry information) as secondary references under the study identifier of the excluded study.

Data from clinical trials registers

If data from included trials are available as study results in clinical trials registers, such as ClinicalTrials.gov or similar sources, we will make full use of this information and extract the data. If there is also a full publication of the study, we will collate and critically appraise all available data. If an included study is marked as completed in a clinical trial register but no additional information (study results or publication, or both) is available, we will add this study to the 'Characteristics of studies awaiting classification' table.

Assessment of risk of bias in included studies

Two review authors (BR, BB) will independently assess the risk of bias for each included study. We will resolve disagreements by consensus or by consulting a third review author (MIM). If adequate information is not available from the study publications, study protocols, or other sources, we will contact the study authors for more detail to request missing data on items relating to risk of bias.

For randomised controlled trials (RCTs) we plan to use the second version of the Cochrane risk of bias tool (RoB 2); for non‐randomised clinical studies we plan to use the Cochrane 'risk of bias in non‐randomized studies of interventions' (ROBINS‐I) tool (Cochrane Methods Resoures). However, we do not expect to find clinical studies for our review topic. For all other types of included studies we will try to address risk of bias in a meaningful and transparent way, if possible using appropriate instruments described in the literature.

Dealing with missing data

We will try to obtain missing data from the authors of included studies.

Data synthesis

We do not expect data suitable for meta‐analysis. If possible, we will perform statistical analyses according to the statistical guidelines presented in the Cochrane Handbook for Systematic Reviews of Interventions (Deeks 2019). In case statistical analyses are not possible or meaningful, we will try to present data in a highly structured tabulate format which will be stored online in an open repository (Zenodo.org).

Subgroup analysis

We expect the following characteristics to introduce heterogeneity, and we plan to investigate the following subgroups:

• insulin type;

• storage temperature (depending on data);

• usage time (depending on data);

• injection device.

Sensitivity analysis

Should meta‐analysis be possible, we plan to explore the influence of important factors on effect sizes, by performing sensitivity analyses in which we restrict the analyses to the following:

• published studies;

• studies with low risk of bias;

• large studies, to establish the extent to which they dominate the results.

Certainty of the evidence

For clinical studies we will present the overall certainty of the evidence for each outcome specified below, according to the GRADE approach, which takes into account issues related to internal validity (risk of bias, inconsistency, imprecision, publication bias) and external validity (such as directness of results). Two review authors (BR, BB) will independently assess the certainty of evidence for each outcome. We will resolve any differences in assessment by discussion or by consultation with a third review author (MIM). We will use GRADEpro GDT software and will present evidence profile tables as an appendix.

If meta‐analysis is not possible, we will present the results in a narrative format in the summary of findings table. We will justify all decisions to downgrade the certainty of the evidence by using footnotes, and we will make comments to aid the reader's understanding of the Cochrane Review when necessary.

Summary of findings table

We will present a summary of the evidence in a summary of findings table. For clinical studies this will provide key information about the best estimate of the magnitude of effect, in relative terms and as absolute differences for each relevant comparison of alternative management strategies; the numbers of participants and studies addressing each important outcome; and a rating of overall confidence in effect estimates for each outcome. We will create the summary of findings table using the methods described in the Cochrane Handbook for Systematic Reviews of Interventions (Schünemann 2019).

According to our PICOT setting, interventions presented in the summary of findings table will be the storage of human insulin above or below manufacturers' recommended temperature storage range or advised usage time, or both. The comparators will be storage of human insulin according to manufacturers' recommended temperature storage range or advised usage time, or both

We will report the following outcomes, listed according to priority:

• potency/bioactivity parameters of human insulin;

• diabetic ketoacidosis;

• hypoglycaemic episodes;

• adverse events;

• health‐related quality of life;

• HbA1c;

• fasting blood/plasma glucose.

Appendices

Appendix 1. Search strategies

PMIDs (PubMed‐IDs) of 24 relevant references used to develop the search strategies (Ovid syntax)
(5039832 or 5668013 or 9811506 or 11044282 or 15111554 or 16101301 or 16969698 or 18463345 or 19217734 or 19797814 or 20687866 or 20920435 or 22538135 or 24246357 or 24876429 or 27472257 or 30815830 or 31009254 or 31994414 or 32281880 or 33259116 or 33534816 or 12941735 or 29114035).ui. 

Cochrane Central Register of Controlled Trials (Cochrane Register of Studies Online)

1. MESH DESCRIPTOR Drug Stability

2. MESH DESCRIPTOR Drug Storage

3. MESH DESCRIPTOR Tropical Climate

4. MESH DESCRIPTOR Thermodynamics

5. #1 OR #2 OR #3 OR #4

6. MESH DESCRIPTOR Insulin

7. insulin*:TI,AB,KY

8. #6 OR #7

9. #5 AND #8

10. ((temperature* OR stability OR storage OR thermostab* OR heat*) ADJ12 insulin*):TI,AB,KY

11. #9 OR #10

 

MEDLINE (Ovid)

1. (Drug Stability/ or Drug Storage/ or Tropical Climate/ or Thermodynamics/) and insulin*.mp.

2. ((temperature* or stability or storage or thermostab* or heat*) adj12 insulin*).tw.

3. 1 or 2

 

CINAHL (EbscoHost)

1. MH "Drug Stability"

2. MH "Drug Storage"

3. S1 OR S2

4. MH "Insulin"

5. TI (insulin*) OR AB (insulin*)

6. S4 OR S5

7. S3 AND S6

8. TI ((temperature* OR stability OR storage OR thermostab* OR heat* OR thermodynamic* OR climate*) N12 insulin*) OR AB ((temperature* OR stability OR storage OR thermostab* OR heat* OR thermodynamic* OR climate*) N12 insulin*)

 

Science Citation Index Expanded (Web of Science)

AB=(("tropical climate" OR thermodynamic* OR temperature* OR stability OR storage OR thermostab* OR heat*) NEAR/12 insulin*) OR TI=(("tropical climate" OR thermodynamic* OR temperature* OR stability OR storage OR thermostab* OR heat*) NEAR/12 insulin*)

 

WHO Global Index Medicus

Title: (insulin* AND (tropic* OR thermodynamic* OR temperature* OR stability OR storage OR thermostab* OR heat* OR hot OR thermal OR environment* OR climate*))


WHO ICTRP Search Portal (Standard search)

insulin* AND (tropic* OR thermodynamic* OR temperature* OR stability* OR storage* OR thermostab* OR heat* OR climate* OR thermal*)

 

ClinicalTrials.gov (Advanced search)

Title/Acronym: insulin AND (tropical OR tropics OR thermodynamic OR thermodynamics OR temperature OR temperatures OR stability OR storage OR thermostability OR heat OR hot OR thermal OR environment OR environmental OR climate OR climates)

Contributions of authors

All review authors contributed to, read and approved the final protocol draft.

Sources of support

Internal sources

• No sources of support provided

External sources

• World Health Organization (WHO), Other

  This review will be funded by WHO.

Declarations of interest

Brenda Bongaerts: none known

Maria‐Inti Metzendorf: none known

Bernd Richter: this review will be funded by the World Health Organization (WHO)

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