= Emerging. More than 5 years before clinical availability. (17.55%, 2019 US)
= Expected to be clinically available in 1 to 4 years. (42.72%, 2019 US)
= Clinically available now. (39.74%, 2019 US)

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MSACL 2019 US : Girtman

MSACL 2019 US Abstract


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Topic: Troubleshooting

The Challenges Associated with the Development of a Clinically Viable Quantitative Insulin Analog Assay

Adam Girtman (Presenter)
Mayo Clinic

Authors: Adam B. Girtman, Stefan Grebe, and Ravinder J. Singh
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN

Short Abstract

Insulin analogs have been genetically or chemically manipulated to behave differently in the body, depending on the patients’ needs, either as rapid-acting or long-acting insulin. Current immunoassays used to detect insulin have been designed to detect human insulin, and depending on the assay manufacturer, the immunoassays ability to detect modified insulin analogs varies greatly. We set out to develop an LC-High Resolution Accurate Mass (HRAM) clinical assay that is able to quantitatively determine seven different insulin analogs. Through many experiments, trials and tribulations we have gathered much information, which could be useful for other laboratories future insulin assay development.

Long Abstract

Problem

Insulin analogs have been genetically or chemically manipulated to behave differently in the body, depending on the patients’ needs, either as rapid-acting or long-acting insulin. Current immunoassays used to detect insulin have been designed to detect human insulin, and depending on the assay manufacturer, the immunoassays ability to detect modified insulin analogs varies greatly. We set out to develop an LC-High Resolution Accurate Mass (HRAM) clinical assay that is able to quantitatively determine seven different insulin analogs. Through many experiments, trials and tribulations we have gathered much information, which could be useful for other laboratories future insulin assay development.

Method Information

Method:

1. 500mcL of human serum

2. 50mcL of bovine insulin (1nM), used as an internal standard

3. Immunoaffinity capture onto antibody packed tip

4. Elute with 105mcL 33% ACN, 0.4% TFA

5. Inject 45mcL on a 2.1x100 3.5u diphenyl column

6. Each insulin is quantitated off their ‘+5’ charge state

accurate mass, and confirmed via unique transitions

Troubleshooting Steps

Many improvements were made throughout assay development by using inter-assay precision as a barometer for each experiment. Insulin and the different analogs tested provided multiple challenges as each of them has different molecular properties.

The structure and sequences of the insulin analogs provided many difficulties during the development process. For example, human insulin and insulin lispro, have identical masses and a nearly identical amino acid sequence, with only two amino acids flipped (P and K) near the C-terminus of the B chain. Using the ‘+5’ charge state, the transitions were identical and were unable to differentiate between human insulin and lispro without chromatographically separating on the LC column. Using a shallow gradient, 20-29%, over the first seven minutes of the method, both human insulin and insulin lispro were separated on column.

New column equilibration proved troublesome, due to insulins propensity to adhere to many open sites. By flushing the column for two hours with a 80:20 water:acetonitrile plus 0.05% bovine serum albumin (BSA) solution, followed by one hour with water and finishing with one hour of 80:20 water:acetonitrile; the column was reproducibly equilibrated over several different lots.

Insulin detemir concentration, with its C14 myristic acid fatty chain, would decrease over time following elution from the antibody tips. This was due to the hydrophobic peptides sticking to plastic collection plates. By changing from plastic collection plates to glass inserts the precision decreased at least five fold for detemir and all other analogs, into an acceptable range of <10% CV.

The elongation of both our immunoaffinity capture and elute dramatically increased our precision. By changing the capture step from five hundred to eight hundred and doubling the elution to two hundred aspiration and dispenses, an extra ninety minutes was added to the run, but precision was cut in half from around 10%CV to 5% for all analogs.

Outcome

Many different experiments and trials have led to a successfully developed clinical assay that separates out seven insulin analogs down to a lower limit of quantitation (LLOQ) of 2.5mcIU/mL. The insulin analogs that are currently monitored: human insulin, insulin aspart, insulin detemir, insulin glargine metabolite 1, insulin glargine metabolite 2, glulisine, insulin lispro.


References & Acknowledgements:


Financial Disclosure

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