Antibody Storage and Antibody Shelf Life

Antibody storage 'shelf life' may range from several weeks to many years depending on both the intrinsic properties of the antibody and the storage conditions. A number of diagnostic antibodies have been shown to maintain their functionalities after 12-26 years of storage at 4°C [2]. However, optimal conditions for storage are unique to each antibody. For example, Laskowski TJ et al found that their 17-color flow cytometry panel antibody mixes remained stable for up to 15 days at 4°C while CyTOF antibody cocktails had a maximum storage time of 3 days at 4°C [3]. Nevertheless, some general guidelines can be applied to increase the shelf-life of your antibodies in storage. Antibodies must be stored at appropriate temperature and pH ranges, and frequently, in the presence of concentrated (~1 M) substances like glycerol or sucrose, in order to retain activity and prevent aggregation. Many commercially available antibody reagents contain one or more additives discussed below that are intended to increase the stability of the antibodies during long-term storage. Table 1 summarizes common antibody storage conditions and other characteristics.

aqueous, 4°C 25-50% glycerol or ethylene glycol, -20°C frozen at -20 - -80°C or in liquid nitrogen lyophilized*1 typical shelf life 1 month 1 year years years antibody concentration 1-5 mg/ml 1-5 mg/ml 1-5 mg/ml 1-5 mg/ml carrier proteins for dilution BSA BSA BSA no sterile or antibacterial requirement yes usually no no antioxidants usually; 2-ME, DTT*2 usually; 2-ME, DTT*2 no no fluorescence conjugation protect from light protect from light protect from light protect from light pH value 7.2-7.6 7.2-7.6 none none metal chelator EDTA EDTA no no multiple uses of single aliquot yes yes no; freeze-thaw cycles degrade antibodies non-applicable Table 1. Antibody storage condition. *1 discussed in detail below. *2 methionine can also be considered. . Antibody storage condition. *1 discussed in detail below. *2 methionine can also be considered.

Many reagent suppliers also provide PBS-only antibodies (without stabilizing additives or carrier proteins such as BSA, or sodium azide, or glycerol), which can be used in conjugation reactions with dyes and enzymes, or in functional/cell assays, or in live-cell imaging experiments. These PBS-only antibodies tend to be supplied at higher concentrations.

Storage Conditions

Laboratory equipment: refrigerators and storage vials

When storing antibodies below zero, it is important NOT to use frost-free refrigerators. Many household refrigerators have a frost-free feature that occasionally performs freeze-thaw cycles to prevent frost formation. These repeated freeze-thaw cycles can substantially reduce the functional shelf-life of your antibodies.

Many antibodies intended for labeling experiments are conjugated with fluorescent dyes. Light exposure can bleach these dyes and dramatically reduce their fluorescence. Such conjugated antibodies must be protected from light exposure by, for example, storing in dark vials or in vials covered with aluminum foil.

Temperature

Chemical reactions such as oxidation and proteolytic degradation of proteins do occur at moderate temperatures; however, the frequency of these reactions is much greater at higher temperatures. Antibodies are generally stored at ≤ 4℃ in clean and sterile glassware or polypropylene tubes. Storage at room temperature often leads to antibody degradation and/or inactivity, usually resulting from microbial growth. For short term storage (1 day to a couple weeks), well prepared antibody stock solutions may be stored at 4°C without significant loss of activity. Under poor storage conditions of 40° for 2 weeks at a typical formulation pH of 6.0, aspartate and asparagine residues within degradation “hotspots” of the variable F v region were found to be modified in as many as 39% of antibody molecules [4].

Cryoprotectants: glycerol and ethylene glycol

Ice crystal formation can destroy protein structure, and thus render antibodies ineffective. Cryoprotectants such as glycerol and ethylene glycol prevent ice crystal formation by inhibiting hydrogen bonding between water molecules, and thus decrease the freezing points of aqueous solutions. The actual freezing point is dependent on the properties and concentration of the cryoprotectants (see Table 2).

10 20 30 40 50 60 70 80 90 100 glycerol -2 -5 -10 -16 -22 -34 -38 -19 -2 17 ethylene glycol -4 -7 -15 -23 -34 -48 -51 -45 -29 -12 Table 2. Glycerol and ethylene glycol freezing points (in Celsius) at different concentrations (in percentage %) in water. . Glycerol and ethylene glycol freezing points (in Celsius) at different concentrations (in percentage %) in water.

(Note: ethylene glycol is toxic, and must be handled with care.)

When the storage temperature is below the freezing point for a cryoprotectant-antibody solution, the solution will solidify. However, instead of ice crystal formation, vitrification typically occurs, as in the cryopreservation of cells/embryos with DMSO. During vitrification, the solution solidifies without crystal formation and thus the structural integrity of the antibodies is maintained.

For increased stability, glycerol or ethylene glycol can be added to a final concentration of 50% and the antibody can then be stored at -20°C. This antibody solution should be stored in small working aliquots, for example, 25 ul [5], so they are subjected to fewer freeze-thaw cycles that can denature the antibodies.

Since glycerol can be contaminated by microbes, it is critical to use sterile glycerol preparations when using it for antibody storage.

Figure 1. Sodium azide at different concentrations inhibits the growth of Gram-negative bacteria. From [ Sodium azide at different concentrations inhibits the growth of Gram-negative bacteria. From [ 1 ].

Sterilization and antimicrobial compounds

Antibody preparations should always be sterilized through filtration using a 0.45-micron filter and must be handled aseptically to prevent microbial contamination. Anti-microbial agents such as sodium azide (NaN 3 ) at a final concentration of 0.02-0.05% (w/v) or thimerosal at a final concentration of 0.01 % (w/v) or ProClin 300 at 0.02% can also be used to inhibit microbial growth.

The most commonly used of these antimicrobial agents is sodium azide, which is toxic to most organisms, including humans. Most gram-negative bacteria are well controlled by sodium azide (see figure 1) [6] ; however, many gram-positive bacteria (streptococci, pneumococci, lactobacilli) are resistant to sodium azide (see figure 2) [1, 7, 8]. Sodium azide inhibits cytochrome oxidase in the mitochondrial electron transport chain and induces apoptosis [9]. Antibodies in sodium azide solution should NOT be directly used in living cells or in in vivo studies. In addition, sodium azide interferes with most conjugation reactions, specifically amine group-dependent conjugations. If conjugations is to be performed sodium azide can be removed relatively easily through gel filtration or dialysis, for example, DiaEasy Dialyzer MWCO 12–14 kD from BioVision [10].

Figure 2. Sodium azide fails to inhibit the growth of Gram-positive bacteria. From [ Sodium azide fails to inhibit the growth of Gram-positive bacteria. From [ 1 ].

Thimerosal contains mercury, and is very toxic by inhalation, ingestion, and in contact with skin. It may also cause allergic reactions among a certain population of people. Thimerosal was used as a preservative for medicines and vaccines until it began being phased out in 1999 due to concerns about potential toxicity.

The active ingredients of ProClin 300 are 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, inhibitors of Kreb cycle enzymes α-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH dehydrogenase. According to Sigma, one of its suppliers, it presents no health hazards, toxicology problems, or disposal issues at recommended usage levels (0.03 – 0.05%), and thus is considered a safe alternative to sodium azide and thimerosal.

Protease inhibitors

Antibody proteolysis by proteases can be an important issue for storing ascitic fluid and serum preparations since both preparations contain proteases. Typically cold storage (≤ -20° for long term and 4° for short term) is sufficient to prevent significant proteolytic degradation of the antibody. However, protease inhibitor cocktails, available from several suppliers, may be added to the antibody solution if proteolytic cleavage becomes a problem.

Antibody concentration

Dilute antibody solutions are more prone to inactivation and physical losses as a result of low-level non-specific binding to the storage vessel surfaces. Thus, it is advisable to keep antibody concentration high (e.g. >1 mg/mL) during storage.

If the concentration of an antibody is low, stabilizer proteins (sometimes referred to as carrier or filler proteins), such as purified bovine serum albumin (BSA) or gelatin, can be added to a final protein concentration of 1-5 mg/ml (0.1-0.5%). These stabilizer proteins competitively inhibit/reduce surface tension and non-specific absorption to storage tube and pipette surfaces. These added proteins can also help reduce proteolysis of antibodies.

Dry ice during storage and transport

Research has found that use of dry ice during protein storage and transport can cause acidification of the storage solution and potentially protein aggregation (with or without precipitation), especially with acidic proteins (those proteins with pI less than 7) [11]. Polyclonal IgG tends to be acidic, with a pI range of 4.7-7.5 [12]. Gas-sealed vials and/or gas-sealed plastic bags (for bagging the vials) should be used to minimize CO 2 reaching the antibody solution. Additionally samples should be re-equilibrated in a -80°C freezer for a couple of days. Dri-Shield Moisture Barrier Bags from 3M, Thermo Scientific™ Nunc™ Cryoflex products, and IMPAK bags like 0203PM56OZETN and 05MP081OZE can be useful.

Antioxidants

The effect of oxidation on antibody molecules has been extensively studied, especially for therapeutic antibodies (e.g., [13] ). Several residues of the antibodies, especially methionine and tryptophan, have been found to be susceptible to oxidation in the presence of UV light, elevated temperatures or oxygen radicals [14-17], and oxidation of these residues can alter both the stability and the function of the antibodies.While oxidation does not generally present a problem with antibodies used in research, there are antioxidants, such as 2-ME or DTT, that can slow this oxidation. Recent research suggests free methionine may be a particularly useful antioxidant for protein storage [18].

Lyophilization

Freeze-drying (lyophilization) is the method of choice for long term storage of monoclonal antibodies because lyophilized antibodies are much more stable than they are in solution. Lyophilization involves drying antibodies at a very low temperature, reducing the damage to the products and retaining the molecular integrity. It extends the shelf life of antibodies, substantially eases the shipping temperature requirement and preserves their chemical and biological properties. Lyophilized antibodies are stable for 3-5 years without losing activity if stored at -20°C or below. Generally, the antibodies should be stored lyophilized until they are needed and reconstitution performed shortly before use. Lyophilized antibodies can be reconstituted by adding deionized or distilled water and inverting the container 5-6 times at room temperature. The reconstituted antibody can be stored for several weeks at 2-8°C or for up to 1-2 years at ≤ -20°C.

Antibody stability in a dry solid state is sensitive to pH [19], which plays a dominant role in determining the physical stability of the IgG1 in the lyophilized state, with pH 5.0 being the most stable. There are more aggregates and more secondary/tertiary structure changes at lower pH [19]. Thus buffer salts are generally required in a protein formulation to control the pH and minimize protein degradation during freeze-drying. Residual moisture content also plays a key role in maintaining the stability of the antibodies. Chang et al. found that optimal stability of the pure IgG1 antibody was at a water content of 2%-3% [20]. The lowest water content is not necessarily the optimal condition, and the residual moisture should be optimized during formulation development.

Proteins undergo denaturation, often forming intermolecular β-sheet structures, upon lyophilization in the absence of stabilizers. Different stabilizers such as sugars (sucrose and trehalose are the most used stabilizers) or polyols (glycerol and sorbitol) are normally added to the formulations to protect monoclonal antibodies against degradation during lyophilization and storage. The presence of sucrose in a formulation can help preserve the native structure of the protein in the solid state and inhibit physical instability during long-term storage [20]. Enhanced protein stability is imparted by the amorphous mannitol due to the inability of mannitol to crystallize during freeze-drying, potentially through hydrogen bonding interaction of the polyol to protein sidechains. Sugars in combination with polyols are more effective at preventing aggregation than sugar or polyol alone, as evidenced by the disaccharide/mannitol formulations maintained native structure better than mannitol only formulations. Addition of a small amount of sorbitol to a sucrose-based formulation resulted in greater retention of native structure and improved stability [20].

The level of stabilization afforded by sugars or polyols generally depends on their concentrations. Increasing sugar/polyol concentration to a certain level may eventually reach a limit of stabilization or even destabilize a protein during lyophilization, so a specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody. Many stability studies are performed using iso-osmotic concentrations of sugars (e.g., 275 mM sucrose or trehalose). Jeffrey et al. found a sugar-to-protein molar ratio of 360:1 was sufficient to provide storage stability of rhuMAb HER2, and the sugar concentration was 3-4 fold below the iso-osmotic concentration typically used in formulations [21]. The level of stabilizers used for protein protection during lyophilization depends on the formulation composition, concentration and physical properties of the stabilizer, and its compatibility with the protein. Long-term storage for antibodies at room temperature or above may be achieved by lyophilization via proper selection of the molar ratio and sugar mixture.

The mechanism of stabilization by sugars during drying is either that sugars produce a glassy matrix to restrict mobility (glass dynamics mechanism) and/or act as a water substitute (water substitute mechanism). Most studies supported the latter mechanism. The interaction between water and proteins is critical to the conformational stability of the proteins. When water is removed during drying, the stabilizers can form hydrogen bonds with the protein, as water molecules do, thereby preserving the native protein structure during the lyophilization process.

Lyophilization is a good choice to achieve the desired long term storage for antibodies at room temperature. However, problems still exist. Proteins can become unstable during lyophilization processes and/or long term storage. Lyophilization is a method with poor efficiency, high energy consumption and large investment in equipment. There is no simple, universal protocol in formulating antibodies, and trial-and-error is still required on most occasions.

Light

Aromatic amino acid residues in proteins can absorb UV-light, and light was shown to induce conversion of Trp to Gly and Gly hydroperoxide in IgG1 [22]. Chemicals, such as detergents like Tween 80, in antibody solutions, may introduce additional photosensitivity to antibodies [23]. Additionally, some antibodies are conjugated to fluorescent labels that can quickly bleach with light exposure. Thus prolonged exposure of antibody products to light (especially UV light) is not advisable [24].

Mechanical stresses: vortexing, shaking, and vial-dropping

Proteins, including antibodies, can aggregate and degrade when subjected to mechanical stresses incurred during vortexing, shaking and vial-dropping [25-27]. This is at least partially due to the cavitation and exposure of proteins in the air [27]. For this reason it is best to treat antibody samples relatively gently. For example, lyophilized antibodies can be reconstituted by gentle inversion rather than vortexing.

Polyclonal Antibodies

Very little loss of activity may occur when serum is directly stored for a decade at ≤ -20°C. However, once the polyclonal antibody is purified, some loss of activity occurs slowly over the years. It also seems that glycerol may not be necessary for storage at -20°C for years or even decades if the antibodies do not go through repeated freeze/thaw cycles. However it is important that the stored polyclonal antibodies should be in high concentration.

Monoclonal Antibodies

Monoclonal antibodies can be stored at -20°C in 50% glycerol.

It is also reported that monoclonal antibodies can be stored under saturated ammonia sulfate as pellets at 4°C or -20°C for many years without loss of activity, bacterial outgrowth or oxidation.

Lyophilization (freeze-drying, or drying from the frozen state) provides an alternative method of stabilizing antibodies that are not freeze-labile. In most situations, freeze-dried proteins can be stored at -20°C.

Conjugated antibodies

Conjugated antibodies, especially those with fluorescent labels, should be stored in dark containers or covered in aluminum foil.

Enzyme-conjugated antibodies

Alkaline phosphatase and other enzyme conjugates are particularly sensitive to freezing, and should in general be stored at 4°C for short term after conjugation .

Antibody conjugates are best stored at -20°C with glycerol or ethylene glycol at a final concentration of 50% for the long term. Although some enzyme conjugates may be stored at -20°C without cryoprotectants, frozen stocks must be single-use aliquots to prevent repeated freeze-thaw cycles.

HRP-conjugated antibodies can be stored in 20-30% serum from horse, adult bovine, calf, or rabbit, or in one of many commercial stabilizers or in a combination of both (Oded Babai, Savyon Diagnostics, Israel).

Fluorescence-conjugated antibodies

As with any reagents conjugated with fluorescent tags (not just antibodies), fluorescence-conjugated antibodies must be protected from light. Fluorescence can photobleach when exposed to light. Exposure to light may render poor performance and vial-to-vial variation. Fluorophore-conjugated antibodies also should be stored at 4°C and should never be frozen. For example, BD Biosciences issued a rare recall of fluorophore-conjugated antibody reagents due to high-intensity lighting in its warehouse in 2017.