Physical state of l-histidine after freeze-drying and long-term storage

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Abstract

Liquid samples of l-histidine of varying pH values and mixed with salt, metal ions, polysorbate 80 and sucrose have been analysed by differential scanning calorimetry to evaluate the influence of these additives on the glass transition temperature and crystallisation of l-histidine during freezing and thawing. l-Histidine solutions of varying pH were freeze-dried with and without a thermal cycle and the physical state of the freeze-dried cakes, following long-term storage, were studied by powder X-ray diffraction. Amorphous l-histidine crystallised when it was exposed to moisture, and the identity of the crystalline materials is reported. The crystallisation of l-histidine during freezing and thawing is dependent on the pH of the solution and is shown to be at a minimum at pH 6, which coincides with the pKa of the imidazoline function. Sucrose inhibited the crystallisation of l-histidine during thawing, while sodium chloride or polysorbate 80 did not. The addition of metal ions (Ca2+ and Mg2+) up to 10% (w/w) did not depress the glass transition temperature significantly, while the addition of Zn2+ increased it. The physical state of l-histidine after freeze-drying is shown to be dependent on both the pH of the solution and the freezing cycle. The risk of crystallisation of amorphous l-histidine is low if the freeze-dried material is protected from moisture.

Introduction

Protein drugs are generally chemically and physically unstable in solution and freeze-drying is frequently used to obtain an acceptable shelf life (MacKenzie, 1966, MacKenzie, 1977; Pikal, 1990). Sugars and/or amino acids are often included in the formulation to prevent inactivation during freeze-drying and to stabilise the protein during long-term storage. Sugars and amino acids protect the protein by preferential exclusion during freezing and by glass formation and/or by functioning as a water substitute in the dried state (Carpenter and Crowe, 1989; Franks et al., 1991; Arakawa et al., 1992). In contrast to sugars, amino acids, in addition to their stabilising properties, may also function as buffers. l-Histidine has recently been shown to function as both buffer and stabiliser in freeze-dried formulations of recombinant factor VIII (Österberg et al., 1997) and recombinant factor IX (Bush et al., 1998). It is a basic amino acid often found at the active site in enzymes and in the coordination of metal ions in metalloproteins. The specific properties of l-histidine reside in the imidazoline function, which possesses both basicity and π-electron acceptor capability (Sundberg and Martin, 1974). The imidazoline function confers good buffer capacity in the pH range 5–7 (Fig. 1), which is often a suitable pH range for many protein drugs. l-Histidine forms strong complexes with certain metal ions such as Cu2+, Zn2+ and Fe2+, but simple salts with the alkali metals (Na+, K+, Ca2+and Ba2+) (Greenstein and Winitz, 1961). The latter fact is especially important in the formulation of protein drugs that require free calcium ions in the buffer for stability reasons (e.g. factor VIII). Trace levels of metal ions such as copper and iron are often present in buffer salts. These ions can often facilitate the oxidation of proteins (Lamfrom and Nielsen, 1970; Shihong et al., 1993). Since l-histidine forms strong complexes with these ions, it may also function as an antioxidant. The solubility of l-histidine in water is 41.9 mg/ml at 25°C and is sufficient for proper buffering and to allow it to function as a non-crystallising (amorphous) stabiliser for many protein drugs. In this study the freezing/thawing behaviour of l-histidine of varying pH in the presence of sodium chloride, metal ions and sucrose was studied by means of differential scanning calorimetry (DSC). l-Histidine solutions of varying pH were freeze-dried with and without a thermal cycle. The freeze-dried cakes were examined with powder X-ray diffraction after long term-storage and after moisture exposure. The exposure to moisture induced crystallisation and the identity of the crystalline materials is reported.

Section snippets

Materials

l-Histidine used for the freeze-drying experiments conformed to the requirements laid down in DAB and USP. l-Histidine (SigmaUltra), l-histidine monohydrochloride monohydrate, sucrose (SigmaUltra), and sodium chloride (SigmaUltra), used for DSC studies and as references for the powder X-ray experiments, were from Sigma (Sweden). Magnesium chloride (ACS reagent grade, ICN Biomedicals), calcium chloride dihydrate (ACS reagent grade, Acros) and Tween 80 (Polysorbate 80) were obtained from Chemicon

Results and discussion

The selection of buffer for a protein formulation is very important and several factors have to be considered. The buffer must have low local and systemic toxicity and be compatible with the active protein and other essential ingredients (e.g. metal ions). It must also be chemically stable and the pKa should preferably be close to the formulation pH in order to give good buffer capacity. The latter requirement also implies that the extent of ionisation of the buffer varies considerably around

Conclusions

l-Histidine may be regarded as a multifunctional protein stabiliser since it can function as a buffer and a metal ion scavenger and stabilise the protein in an amorphous phase. When formulating proteins with l-histidine, it is important to consider the pH value with regard to both possible crystallisation of l-histidine and its influence on the Tg′. Metal ions such as Ca2+ and Mg2+ can be added to l-histidine without significant depression of Tg′. The risk of crystallisation of amorphous l

Acknowledgements

We gratefully acknowledge the assistance of Johan Andersson, Astra Arcus, who performed the moisture balance studies.

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