Reagent Formulation and Shelf Life Evaluation | Products | Immunology

In this article we will discuss about:- 1. Introduction to Reagent Formulation 2. Classification of Reagent Formulation 3. Shelf Life of a Product.

Introduction to Reagent Formulation:

Formulation is a mixture or substance prepared as per a formula. Formulations are made for a particular application and normally are hyperactive than its individual components when used alone. The quality of an assay system largely depends on the source and preparation of reagents. In the commercial kits, the reagents are reconstituted before use.

Extra care should be taken while reconstituting the freeze dried reagents. Freeze dried reagents are packed at negative pressure and so they should be opened very carefully allowing air to slowly enter the vial. Then such reagents are reconstituted with correct solvent using calibrated pipettes at the recommended tempera­tures.

The solvent and reagent is then mixed carefully to avoid foam formation (which may lead to denaturation of protein reagents). We should strictly follow the manufacturer’s instructions while preserving the reconstituted reagents. Some general guidelines have been given by Blockx and Martin, 1994 for this purpose.

Classification of Reagent Formulation:

Reagent formulation can be divided into two types:

These are:

(1) Liquid Formulations.

(2) Solid Formulations.

(1) Liquid Formulations:

A number of reagents such as coating buffers, blocking buffers, incubation buffers washing buffers, enzyme label, substrate buffers, chromophores and stop reagents can be formulated and supplied as liquid reagents in commercial immunodiagnostic kits. Most of the reagents are aqueous solutions.

Therefore, the stability and performance of these reagents in the immunoassays depend on the quality of water used. Untreated water contains a no. of impurities including inorganic, organics, dissolved gases, suspended solids, colloids, microorganisms and pyrogens. Deionization and reverse osmosis are used for preparing a quality source of water for laboratory use.

(i) Deionization and reverse osmosis prepares water for laboratory use.

(ii) Cation and anion exchange resins removes ionizable substances.

(iii) Ion exchange resins or activated carbon filters remove organic and colloid matter.

The ideal water quality for making reagents and calibrations should have resistivity = 10 – 18 MΩ/cm at 25°C; pH – 7.0; Total Organic Content (TOC) – 20 – 100 ppb. Most of the immunoassays give equally good results with water having resistivity not lower than 5 MΩ/cm at 25°C and Total Organic Content (TOC) of less than 2 ppm. Immunodiagnostic kits use many important solutions.

Formulation of some of the solutions is discussed below:

(i) Coating Buffers:

a. Coating buffers should have pH at least 1 – 2 units higher than the pI of the protein.

b. Coating buffers are used for adsorbing antibodies or antigens on the solid surface.

c. Coating buffers ionic strength should be low.

d. Commonly used Coating buffers are Phosphate (pH 7.0 – 7.2) and carbonate (pH 9.2 – 9.4) buffers of 0.02 – 0.05 molarity.

e. Coating buffers should contain only the primary coating molecule of interest to bind to the surface.

(ii) Blocking Buffers:

a. Blocking buffers should also be as pure as possible

b. Blocking buffers should contain the buffer salts and the blocking agent only

c. The blocking agent should not cross react in the later steps of the assay.

d. The blocking agent should not have any affinity for antigen, antibody or the enzyme label.

(iii) Incubation Buffers:

a. Commonly used incubation buffers are phosphate buffer, Tris buffer, borate buffer, barbital, glyine or bicarbonate buffer.

b. Incubation buffers should have a neutral pH value.

c. Incubation buffers should have a low to medium – high ionic strength.

d. Composition of incubation buffers should be such that it ensures the binding of analyte to the antibody.

e. Blocking agent should be one of the constituents of the incubation buffers so as to improve assay performance.

(iv) Wash Buffers:

a. Washing buffers should have a low to medium-high ionic strength.

b. Washing buffers should have a pH value of 7.0.

c. Incorporation of detergents (Tween-20) in washing buffers avoids cross-reactivity problems.

(v) Substrate Buffers:

i. Formulation of substrate buffers are mandated by the enzyme label and the substrate system used in the assay protocol.

ii. The standard protocols should be followed.

(vi) Enzyme and Antibody Labels:

The below mentioned points are important for enzyme and antibody formulations:

a. Formulations should be prepared in phosphate buffered saline of pH 7.0 – 7.2. Cryoprotectant (Polyols, Glycerol, Polyethylene glycol, etc.) should be added to the enzyme.

b. High concentrations of sugars crystallize out when stored for long periods. Therefore, they must be avoided in such formulations.

c. High temperature (35°C and above) must be avoided as it may inactivate/denature the enzyme.

d. Acid and alkaline solutions (pH less than 5 or more than 9) must be avoided as it may inactivate/denature the enzyme.

e. Foaming must be avoided while stirring the enzyme or antibody solutions.

f. Repeated freezing and thawing should be avoided.

g. Certain metal ions can inactivate the enzymes; therefore, millipore water should be used for such preparations.

(vii) Preservatives:

Commonly used preservatives are:

(a) Thimecosal (Merthiolak â):

i. Expensive.

ii. Toxic for disposal as it contains mercury.

iii. Blocks some chromogenic substrates.

(b) Sodium Azide:

i. Hazardous.

ii. Product disposal should be done safely.

iii. Inhibits a number of biological assays.

iv. Inhibits a number of enzymes used in immunoassays.

(c) ProClinTM 300:

i. New biocide for in vitro diagnostic reagents.

ii. It consists of two isothiazolones:

a. 2-methyl-4-isothiazolin-3-one.

b. 5-chloro-2-methyl-4-isothiazolin-3-one.

iii. It is not recommended for freeze dried solid formulations.

iv. It has broad spectrum biocidal activity.

v. Low toxic.

vi. Good compatibility and stability.

vii. Eradicates bacteria, fungi and yeast cells for longer periods.

(2) Solid Formulations:

There are some reagents/solutions which are unstable during the product life span for distribution and use in aqueous solutions. Such reagents come under the solid formulation category. Freeze drying or lyophillization is the method used for preparing solid reagent formulations for use in commercial immunoassay kits.

Freeze-Drying:

i. Provides extended stability and shelf life to the product.

ii. Minimizes chemical decomposition or loss of biological activity.

iii. Provides a sterile and low particulate process.

iv. Facilitates accurate and sterile dosage when carried out in vials.

Freeze drying process consists of three stages:

(i) Freezing.

(ii) Primary drying.

(iii) Secondary drying.

The freeze drying process is as follows:

The freeze drying system itself comprises of six integrated subsystems:

(a) Product chamber.

(b) Condenser.

(c) Heat transfer system.

(d) Refrigeration system.

(e) Vacuum system.

(f) Instrumentation for process control.

Therefore, the freeze drying process is very important for solid reagent formulations in the commercial immunoassay diagnostic kits.

Shelf Life of a Product:

Shelf life is the recommendation of time that products can be stored, during which the defined quality of a specified proportion of the goods remains acceptable under specified conditions of distribution, storage and display or shelf life (stability) is also defined as the time during which the product’s essential performance characteristics are maintained under specific handling conditions.

The quality of a product changes with time and this change in product quality is affected by storage temperature, humidity, packaging protection and product formulation. Shelf life is different from expiration date. Shelf life refers to quality and expiration date refers to safety. A product that has passed its shelf life might still be safe, but quality is no longer guaranteed.

Product expiration dating is the ultimate practical result of determining stability. The experimental protocols commonly used for data collection that serve as the basis for estimation of shelf life are called stability tests. Stability is the inverse of degradation. For commercial immunodiagnostic kits, the reagents are affected primarily by three types of stability.

These are:

i. Chemical or functional stability.

ii. Physical stability.

iii. Bacteriostatic stability.

The desirable length for reagent stability is given below:

Shelf life of a product can be determined using two basic approaches which are as follows:

1. Real Time Stability Testing:

For determining expiration dates, the real-time stability testing acts as the “gold standard”. The test period duration should be long enough to allow sufficient degradation of the product under specified storage conditions. Therefore, in real-time stability testing, a product is stored at recommended storage conditions and monitored until it fails the specification. The real-time stability testing data frequently changes or varies with the change in the testing method.

In real-time stability tests, a product is stored at recommended storage conditions and then monitored for a particular period of time (t_test). At some time, t_s the product will degrade below its specification. The estimated value of t_s can be obtained by modeling the degradation pattern. Testing should be performed at time intervals that encompass the target shelf life and must be continued for a period after the product degrades below specification.

At least three lots of material should be used in stability testing to capture lot-to-lot variation which is an important source of product variability. Real-time product stability testing is necessary to validate stability testing claims for clinical chemistry reagents and reference material.

2. Accelerated Stability Testing:

Accelerated stability testing is commonly used in the development of clinical reagents. It provides an early indication of product shelf life and thereby shortens the development schedule. In accelerated stability tests, the product is stored under stress conditions (such as high temperature, humidity, and pH). Degradation at the recommended storage conditions are predicted by using known relationships between the acceleration factor and the degradation rate.

Temperature is the most common acceleration factor used for chemicals, pharmaceuticals, and biological products because its relationship with the degradation rate is characterized by the Arrhenius equation. Humidity and pH also have acceleration effects but are more complex.

This information is then extrapolated to predict product shelf life and used to compare the relative stability of alternative formulations. A product may be released based on accelerated stability data, but the real-time testing must be done in parallel to confirm the shelf-life prediction.

Following guidelines are used to approximate the shelf life of a product at the desired temperature:

1. 1 month at 50°C is equivalent to 1 year at room temp.

2. 2 month at 50°C is equivalent to 2 year at room temp.

3. 3 month at 37°C is equivalent to 1 year at room temp.

4. 6 month at 37°C is equivalent to 2 year at room temp.

5. 7 month at 37°C is equivalent to 1 year at 4°C.

6. 3 days at 37°C is equivalent to 3-6 months at 4°C.