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University of Groningen

Analytical techniques and formulation strategies for the therapeutic protein alkalinephosphataseEriksson, Hans Jonas Christian

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Publication date:2004

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Citation for published version (APA):Eriksson, H. J. C. (2004). Analytical techniques and formulation strategies for the therapeutic proteinalkaline phosphatase. Groningen: s.n.

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Introduction Chapter 1

1

Chapter 1

Introduction

Chapter 1 Introduction

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1.1 Therapeutic proteins With the ongoing development within biotechnology, molecular biology and the increasing understanding of proteins and their interactions and functions in biological systems, it seems logical that a larger number of new drugs will be derived from this field. Already, quite a large number of therapeutic proteins, e.g. interferons, growth factors, recombinant human DNAse and several vaccines, have found their way to the market and many more are in different stages of clinical trials. Until now, more than 150 pharmaceutical protein medicines and vaccines have been approved by the Food and Drug Administration (FDA). Therapeutic proteins may have some advantages over conventional low molecular weight drugs. For example, their action might be more specific than that of conventional drugs and, possibly, therapeutic proteins are the answer in finding a cure for cancer and other (terminal) diseases. One might think that, since proteins have a more specific effect in biological systems than conventional drugs, side-effects ought to be a considerably smaller problem. This is not always true since therapeutic proteins might enter the body far from their site of action and therefore unwanted side-effects can still take place. However, due to the potentially high potency of therapeutic proteins, smaller doses (a few milligrams) than for conventional drugs (tens or hundreds of milligrams) are needed, to obtain therapeutic efficacy. A major challenge with large molecules such as proteins is the vast number of functional groups that can be the subject of chemical degradation as well as the complex structure of the individual proteins. Generally, it is important to develop efficient techniques for characterization and profiling of these complex protein samples. Even a small conformational change may have a detrimental effect on the function of the protein. Therefore, efforts to correctly characterize as well as creating a suitable and stable dosage form of each therapeutic protein have to be made.


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1.2 Analytical techniques for therapeutic proteins 1.2.1 Assessment of protein purity It is extremely important that pharmaceuticals are as pure as possible, since any impurities may cause severe side-effects during the treatment of patients. The importance of impurity profiling of biotechnological drugs has also been acknowledged by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) [1]. Regulatory authorities demand that the final therapeutic product should be well characterized with defined purity, efficacy, potency, toxicity and so on. The ICH guidelines are divided into four major topics, i.e. the quality, safety, efficacy and multidisciplinary topics (cross-cutting topics which do not fit uniquely into one of the other categories), where the quality topics relate to the chemical and pharmaceutical quality assurance of drugs in general. The quality assurance topics are in turn divided into seven subheadings. These concern stability (Q1), analytical validation (Q2), impurities (Q3), pharmacopoeias (Q4), biotechnological quality (Q5), specifications (Q6) and good manufacturing practice (GMP) (Q7). For biotechnological products the chapter Q5 is especially designated. For traditional small drugs (500-1000 Da) the characterization of purity is usually performed by high-performance liquid chromatography (HPLC). Since peptides and proteins are much larger, they contain many functional groups and in addition their conformation may change, which as mentioned above can alter their function. To cope with the challenge of monitoring the purity and degradation of proteins a number of analytical techniques can be applied. Within this analytical arsenal, separation techniques are indispensable tools for the effective profiling of protein samples. Until lately, high performance liquid chromatography (HPLC) has been the method of choice in protein purity analysis, but more recently capillary electrophoresis (CE) has emerged as a highly efficient alternative and/or complementary separation technique. With respect to detection techniques, mass spectrometry (MS) has gained an important position in the biosciences over the last decade, and its role in protein analysis is rapidly growing. In the following parts of this chapter the use of HPLC, CE and MS for the analysis of pharmaceutical proteins will be highlighted.


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1.2.2 High-performance liquid chromatography Reversed-phase high-performance liquid chromatography (RP-HPLC) RP-HPLC is a separation technique in which the analytes are separated based on hydrophobic interaction between hydrophobic ligands attached to a column support and hydrophobic areas on the protein. Many proteins unfold on contact with the hydrophobic ligands and by being dissolved in an organic solvent of low pH. Consequently, the total number of hydrophobic groups dominates the elution process during RP-HPLC. This means that proteins containing a large proportion of hydrophobic groups will require high concentrations of organic modifier in the mobile phase for elution [2]. Since the total number of hydrophobic groups determines the hydrophobic nature of a protein, it is not unexpected that RP-HPLC of small proteins (less than 50 kDa) is more successful than of larger proteins. At an equal percentage of hydrophobic residues, the organic solvent concentration necessary for protein desorption will increase with protein size. Therefore, high-resolution separations of protein mixtures are commonly performed using gradient elution. It is not necessarily, however, the molecular weight that is the most important factor, but rather the polarity and orientation of the amino acid residues involved in the interaction with the stationary phase. During isocratic elution the best separations are usually achieved when low flow rates are used (typically less than 1 ml/min) while during gradient runs very shallow gradients (about 0.5% per minute) are necessary if high resolution is wanted. This means that one often is faced with a trade-off, i.e. fast analysis or high resolution. The most common stationary phases are microparticulate porous silica derivatized with a silane containing n-butyl, n-octyl or n-octadecyl ligands [3]. The shorter ligands usually give better protein recoveries, and the pore sizes should be in the 100-300 Å range for peptides and 300-4000 Å for proteins. The reason for this is that the solute molecular diameter should be at most one tenth of the pore size to avoid restricted diffusion of the solute and also to allow the total surface area of the sorbent material to be accessible. Another important variable of the reversed-phase material is the particle diameter, where the most commonly used particle diameters are in the range 3-5 µm, but sometimes smaller particles are used. The chromatography of proteins with reversed-phase columns is often problematic compared to RP-HPLC of peptides. In a study by Burton


5

and co-workers [4], 33 different proteins ranging from very hydrophilic to very hydrophobic as well as basic and acidic proteins, were investigated. They found that only six out of the thirty-three proteins yielded acceptable results concerning the quality of the chromatography. The problems increase with the size and/or hydrophobicity of a protein. The reasons are given by the numerous interactions of a protein with components of the mobile phase, the stationary phase, and other sample components and with itself. Low recovery and low resolution because of band spreading, tailing and multiple peak formation are caused by these interactions. Even irreversible binding to the stationary phase often occurs. According to the multifunctional (e.g., acidic) moieties of a protein, a large number of equilibria are responsible for the fact that a protein coexists in many different forms (e.g., more or less protonated). More information about the use of RP-HPLC in the analysis of peptides and proteins can be found in the book "Protein Liquid Chromatography” [5]. The mobile phases in RP-HPLC of proteins are usually acidic, containing trifluoro acetic acid (TFA), phosphoric acid or perchloric acid, while acetonitrile, methanol, and 2-propanol are the most commonly employed organic modifiers. Important factors that also must be considered are column temperature, gradient time, gradient shape and mobile phase flow rate [3]. A more thorough investigation on the influence of organic modifier on peptide and polypeptide retention on octadecyl supports can be found in reference [6]. RP-HPLC is often applied in the field of biotechnology as a means of purification or characterization of biotechnology products. Assessing the purity of such compounds is indeed a challenge and not easy without pure reference substances. Quite recently, a procedure for quantitative analysis of impure proteins without the need for a purified reference sample was developed [7]. In that study tryptophane was used as a calibrant. The reason for this is that tryptophane has a distinct and known extinction coefficient. It was concluded that the quantitation is highly convenient and accurate for proteins containing tryptophane. A few current reviews of the use of HPLC and other chromatographic techniques for the separation of peptides and proteins are available [8, 9]. RP-HPLC is frequently used in the field of clinical chemistry, e.g., for the analysis of urinary proteins. Detecting deviations from the normal pattern means that renal disorders can be diagnosed. This was for example demonstrated by Arai et al. [10], who evaluated the possibility to estimate the ratio of α1-acid glycoprotein and albumin in urin by RP-HPLC using a


6

column packed with poly-porous glass. This ratio can be used to diagnose renal disorders. Human glycoprotein hormones are of great value because they are used as medical preparations, components of diagnostic kits and the substances for immunization in antibody production [11]. In the literature there are many examples where RP-HPLC has been employed in the analysis of glycoprotein hormones [11-13]. The use of HPLC in clinical analysis has also recently been reviewed [14]. One of the most extensively analyzed therapeutic proteins is insulin, probably due to its clinical importance and also because it is a quite small protein, only about 5.8 kDa. Lookabaugh et al. [15] evaluated the quantitation of insulin by two separate methods, RP-HPLC and CE, and found that RP-HPLC demonstrated better precision than the CE method (see Table I). Table I. Comparison between HPLC and CE for the analysis of insulin (n=4). From [15].

Sample Insulin content (mg/ml) HPLC

Found;(RSD%);[% of actual]

CE

Found;(RSD%);[% of actual]

1 2.07 2.077;(0.71);[100.3] 2.019;(1.94);[97.5]

2 1.19 1.172;(0.53);[98.5] 1.219;(5.13);[102.4]

3 4.25 4.309;(0.53);[101.4] 4.160;(3.10);[97.9]

However, in a different study it was concluded that CE and RP-HPLC can give equivalent results and can be used for cross-validation studies [16]. RP-HPLC has also been successfully employed in the monitoring of the stability of insulin. Insulin can form two main degradation products, i.e. the monodesamido-A21-insulin and the monodesamido-B3-insulin that are formed under acidic and neutral conditions, respectively. In Fig. 1 an example of such a separation is shown. In this example a C-18 column and a mobile phase of 0.2 M sodium sulphate buffer (pH 2.3) with acetonitrile was used.


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Fig. 1 Separation of insulin (1) from desamido-A21-insulin (2) and desamido-B3-insulin (3). The left chromatogram was the result of an insulin solution sample stored at 60 °C with shaking, while the right chromatogram is the results of an insulin zinc suspension sample stored at 50 °C without shaking. Clearly, two different degradation products are formed. From [17]. Insulin has also been used as a model compound to evaluate conformational effects in RP-HPLC of polypeptides [18, 19]. The kinetics of the unfolding of insulin was investigated by examination of the dependence of elution profiles on temperature and column residence time. It was also found that one part of the insulin molecule, i.e. the B-chain, dominates the chromatographic behaviour of insulin. The end conclusion of the study was that RP-HPLC can be used to characterize the role of secondary structure in the interactive behaviour of polypeptides and proteins with hydrocarbonaceous ligands. The degradation of insulin has also been successfully monitored by RP-HPLC [17, 20, 21]. Hydrophobic interaction chromatography (HIC) HIC is often used in the separation of proteins. Reversed phase packings are employed as stationary phases, but instead of an organic modifier a salt is used in the mobile phase. The retention in HIC is inversely related to the salt concentration and the salt species can be varied to control the selectivity. HIC is commonly performed on weakly hydrophobic columns (less hydrophobic than those in RP-HPLC) in high salt concentrations of sodium or ammonium sulphate. The proteins are


8

adsorbed to HIC sorbents from high-salt buffers, and the proteins are then eluted with a descending salt gradient, where the proteins elute according to their solubility. The retention of proteins in HIC is sensitive to the hydrophobicity of the stationary phase, which can be altered by both the length and the density of the alkyl side chains of the bonded phase. Even though RP-HPLC is often used in the separation of peptides, it is sometimes necessary to turn to HIC as a separation technique. One such case is when the peptides are likely to precipitate in the mobile phases used in RP-HPLC. This was studied by Alpert [22], who evaluated the use of HIC in the separation of a set of synthetic peptides and small proteins and compared with RP-HPLC. It was found that HIC seems to be broadly applicable to the purification of peptides as well as proteins, and in some cases HIC and RP-HPLC was found to be complementary. For example, a synthetic lipoprotein analogue could not be purified from the impurities using RP-HPLC, while purification by HIC yielded a pure product (data not shown). Size-exclusion chromatography (SEC) In SEC the chromatography depends on the relative molecular size or hydrodynamic volume of the analytes with respect to the size and shape of the pores of the stationary material. To allow the determination of the molecular weight of unknowns the SEC column first has to be calibrated with a set of test compounds with a known molecular mass. SEC is well suited for the measurement of aggregation of proteins, but such measurements are limited to proteins that are linked through covalent or stable non-covalent links. Generally, non-covalently linked proteins dissociate when diluted, which leads to shifts in retention times and altered apparent molecular weights of the aggregates. Since the range of molecular sizes that each SEC column can separate is limited one often needs to use several columns. One way to overcome this dynamic range limit is to place several SEC columns of graded pore size in series. SEC is often used as a confirmatory technique that allows the examination of homogeneity of fractions extracted by other methods. Classical SEC has been used extensively for peptide and protein purification. However, the use of SEC for peptides smaller than about 5000 has been limited, mainly due to the lack of suitable stationary phases. The gels that are commonly used (agarose, dextrane etc.) are often deformed during normal HPLC-conditions. However, with the improvement of stationary phases it is


9

possible that SEC in the future will be more valuable as an analytical tool for peptide and protein analysis. Interestingly, separation based on both a reversed-phase as well as a size-exclusion mechanism may be achieved through the use of high-porosity reversed-phase supports [23]. There are also silica-based SEC-columns, where the porous silica particles have a hydrogel coating, which typically is a few Å thick. The surface of the porous particles is completely covered with this hydrogel, resulting in an interaction between the proteins and the gel phase comparable to that in gel packings. Though these packings do not have the pH range stability of the gels, they can function over a broader pH interval than common silane derivatized silica. SEC has been found to be ideal for the analysis of human growth hormone (hGH) since it can differentiate between monomers and dimers. RP-HPLC, on the other hand, led to the dissociation of the dimer [24]. In a study by Riggin et al. a SEC-method was developed and compared to a rat bioassay. They found that the two methods correlated well but that the SEC-method was much more precise and reliable than the rat bioassay [25]. In Fig. 2 a SEC-chromatogram of biosynthetic hGH is shown, and as can be seen not only the monomer and dimer were detected but also a higher molecular weight species of the hGH.

Fig. 2 SEC of a typical biosynthetic hGH lot. From [25].


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Ion exchange chromatography (IEC) The separation principle in IEC is based on charge but hydrophobic and polar interactions between analyte and stationary phase also play a role in the retention behaviour. The stationary phase (the ion exchanger) interacts with the analytes through electrostatic phenomena, and a buffer with varying pH and/or ionic strength competes with the ion exchanger for molecular binding sites. The two techniques IEC and RP-HPLC often complement each other, i.e. peptides that can not be separated with one technique may be separated by the other. This means that combining IEC and RP-HPLC often enhances the probability that a complete separation of a peptide or protein mixture is achieved. Often, the presence of an organic modifier in the buffer is needed. The reason for this is that otherwise some peptide/protein peaks are eluted slowly due to their hydrophobicity resulting in broad peaks. Many resins used as stationary phases are susceptible to deformation during high flow rates. One way to increase the rigidity of the stationary phases is to bind the ion exchanger to a microparticulate silica gel. One important draw-back with silica gels is that they are not chemically stable, i.e. they cannot withstand pH above 7.5. This limits their practical use since in IEC pH-gradients or pI-gradients are often applied. It has been suggested that, e.g., ion exchangers based on hydrophilic polyether resins may be available in the future, since they seem to possess adequate chemical stability and loading capacity. IEC has been used for separation of rhDNase differing only in the occurrence of deamidation at a single residue. Since rhDNase is glycosylated it is very charge heterogeneous, making it hard to separate intact and deamidated rhDNase using conventional IEC. However, columns packed with polyanionic ligands, such as a tentacle cation exchanger, immobilized DNA and an immobilized synthetic DNA analogue, were found to accomplish separation between deamidated and intact rhDNase [26]. Separation of glycoforms of rhDNase, differing in the extent and position of mannose phosphorylation, has also been obtained by employing an anion exchanger with a polyethyleneimine (PEI) bonded phase. In Fig. 3 results from this study are given [27]. The separations achieved with a common anion exchanger (diethylaminoethyl, DEAE) were clearly not as good (a) as for the PEI-bonded phase (b). IEC is often used for purification of recombinant therapeutic proteins, e.g., for removal of DNA contaminants [28].


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a ba b

Fig. 3 Separation of rhDNase (A=untreated, B=digested with neuraminidase, C=digested with alkaline phosphatase) using a DEAE-column (a) and a PEI-column (b), respectively. The peaks numbered 1-5 were collected as fractions and subjected to further analysis where it was found that fraction 1 contained no phosphate, 2 and 3 contained 1 mol phosphate, 4 contained about 2 mol phosphate and fraction 5 contained 3 mol phosphate per mol of rhDNase The elution buffer was 5 mM 2-(N-morpholino)-ethanesulfonic acid with 1 mM CaCl2 and a gradient going from 50 to 200 mM NaCl at pH 6.0 for (a), and 10 mM N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid with 1 mM CaCl2 and a gradient going from 0.1 to 1.5 M NaCl at pH 7.0 for (b). From [27]. Affinity chromatography Affinity chromatography is sometimes referred to as biospecific interaction chromatography. This is a technique that is based on specific interactions between molecular pairs from biological systems. Examples of ligands that can be used are antigens, antibodies, hormones, cofactors, receptors etc. This means that it is possible to tailor make a column with affinity only for the analyte of interest, i.e. only this is retained on the column and the rest is eluted. At the end an elution buffer, which disrupts the ligand/protein bond, is applied to rinse the analyte from the column and (ideally) obtain a pure product. This can be very cost effective since as much as 50-80% of the total direct production cost of manufacturing a therapeutic product comes from the final steps, i.e. purification and polishing of the product [29]. Therapeutic proteins often contain mixtures of isoforms, which originate from post-translational modifications in the host cell expression system, e.g., glycosylation, sulphation, oxidation etc. [29]. Since some ligands employed in affinity chromatography originate from natural sources themselves they must also be purified, since they may contain host DNA, and viruses and often show lot-to-lot variation. One way to reduce these problems is to use synthetic ligands such as the


12

biomimetic textile dyes. These have many advantages over natural ligands and are frequently used to purify proteins. However, their selectivity is sometimes not good enough and therefore synthetic ligands are designed to suit only the analyte of interest. A review that covers this was recently published [29]. The conclusion was that synthetic ligands are inexpensive, scalable, durable and reusable. The use of affinity chromatography in the industrial purification of plasma proteins for therapeutic use was also recently reviewed [30]. An efficient way to avoid the problems associated with biological ligands in affinity chromatography is to use so called biomimetics as ligands, which are substances that are structurally similar to their in vivo parent molecule, but are more stable or provide better selectivity for separation purposes. One obvious area where biomimetic ligands can be used is in enzyme purification, where the enzyme binds to the ligand. As a matter of fact, there are many examples of enzymes where this has been used, e.g., trypsin, urokinase, alkaline phosphatase, and malate dehydrogenase [31]. The preparation of an affinity chromatography medium for purification of alkaline phosphatase was demonstrated by Guo and co-workers [32]. They used the triazine dyes Cibracon Blue F3GA and Active Red K2BP that were immobilized as affinity ligands on a chemically cross-linked cellulose film. Using this membrane they managed to purify commercially available calf intestinal alkaline phosphatase about 40 times and the recovery was approximately 60%. The purification of calf intestinal alkaline phosphatase and human urine urokinase by purpose-designed affinity ligands attached to Dynospheres XP-3507 was described by Clonis and Lowe [33]. They also compared the purpose-designed ligands with two other reference ligands, i.e. 6-aminohexyl C.I. Reactive Blue 2 and 4-aminobenzyl phosphonic acid that are known to interact with alkaline phosphatase and urokinase, respectively. Their conclusion was that the purpose-designed ligands were much better than the reference ligands in purification and recovery. Roy and Gupta have described the purification of alkaline phosphatase from chicken intestine by affinity chromatography [34]. The purification progress was followed by enzymatic activity assays where a substrate (para-nitrophenyl phosphate) was converted to a coloured product (para-nitrophenol) that was measured spectrophotometrically. In Fig. 4 a repro-duction of results presented by the authors is given.


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0

500

1000

1500

2000

2500

0 1 2 3 4 5 6 7 8 9 10 11 12 13Fraction

Act

ivity

(U)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Protein (mg)

Fig. 4 Purification of alkaline phosphatase from a crude extract of chicken intestine. The squares show the activity and the circles the amount of protein in each fraction. As can be realized from the figure a large amount of protein is eluted in the beginning (fraction 1-6), where also some enzymatic activity can be found. In the later fractions (10-12), a small amount of protein with a relatively higher enzymatic activity is found, indicating that the early fractions were not pure. Reproduced from [34]. Affinity chromatography has also been applied as a means to determine liver- and bone-derived isoenzymes of alkaline phosphatase in serum [35]. A column of wheat-germ lectin conjugated to 7-µm diameter silica particles and an eluent containing N-acetyl-D-glucosamine was used to separate bone and liver isoenzymes of alkaline phosphatase with a peak overlap of about 10%. 1.2.3 Capillary electrophoresis Electrophoresis methodologies are the most commonly used in the separation of proteins, and are the methods of choice for the majority of protein chemists. However, for large-scale procedures (milligram and gram quantities) liquid chromatographic techniques are commonly preferred, since these have larger loading capacity. For purification of small amounts of polypeptides polyacrylamide gel electrophoresis (PAGE) offers the best results. Electrophoretic methods have unsurpassed resolving power and speed and are suitable for the analysis of both hydrophobic as well as hydrophilic peptides and proteins. Capillary electrophoresis (CE)


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techniques, which have seen a tremendous development during the last two decades, are more and more becoming routine techniques in the biotechnology discipline. For example, when several different glycosylated forms of a protein exist in the same fermentation medium and these forms have different therapeutic activity, the need for a detailed analysis of these forms increases as well. This capability is provided by CE techniques, which consequently are used. Since the progress in biotechnology is an on-going process, the application of CE to the analytical problems encountered in this field is also advancing at the same fast rate. The first biotechnology products were produced using Escherichia coli as host and were, consequently, non-glycosylated and free of most post-translational modifications as well. Nowadays, biotechnology products are produced in mammalian cells, and the resulting proteins are much more complicated. These products possess more complex structural features, such as lipidation, phosphorylation, glycosylation and gamma-carboxylation. Identification of these modifications is vital, since any one of them can have an influence on the activity of a therapeutic protein. The differences in properly and improperly modified proteins are usually small when compared to the molecular size of biologically active proteins that commonly are in the range 20-200 kDa. However, the differences usually involve changes in net charge of the protein. The advantage of capillary electrophoretic techniques is that they provide a charge-based separation mode, and can therefore also give some information about the structure of the protein. The application of capillary electrophoretic techniques in biotechnology has recently been reviewed by several authors [36-42], and the application of CE in the clinical laboratory was also recently reviewed [43]. Even though CE has many advantages there are also disadvantages. Capillary zone electrophoresis (CZE) could in a sense be referred to as “anti-chromatography”, since any interaction with the stationary phase, i.e. the capillary wall, is undesired. Peak broadening and loss of separation efficiency can be caused by adsorption of the proteins to the capillary walls. To counter this phenomenon, one can apply dynamic or static coating of the capillary walls. Dynamic coating is achieved by mixing a suitable additive, i.e. polymer, detergent, or polycation to the background electrolyte. The state of the art of dynamic coatings was recently reviewed [44, 45]. Static wall coatings are often more stable and more efficient than


15

dynamic coatings when the goal is to eliminate the electroosmotic flow. Permanent wall coating was recently reviewed [45]. In the review by Roche et al. [41] the history of CE and several different modes of CE are described. In Table II some of the most common modes are outlined. Table II. Different modes of CE separation.

Mode Basis for separation Separation medium Class of molecules

CZE

Charge and size

Low ionic strength buffer

Small molecules

Peptides

Proteins

MEKC

If charged

-charge and size

If neutral

-hydrophobicity and size

Low ionic strength buffer and a micelle-

forming surfactant

Small molecules

Peptides

Proteins

CIEF

Isoelectric point

Acidic (e.g. H3PO4) and basic (e.g. NaOH)

components, linear polymer, and

ampholytes

Proteins

CGE

Chemical gel

-size

Physical gel

- size

Polymerized rigid matrix or replaceable

gel

Replaceable gel

Proteins

dsDNA

ssDNA

Capillary zone electrophoresis (CZE) CZE is the simplest and most popular form of CE. It is applicable for the separation of a diverse array of analytes varying in both size and character. Separation is performed in low ionic strength buffer, and the separation is based on differences in the charge-to-mass ratio of the analytes. Thus, both small molecules and large macromolecular protein complexes can be separated in the same mode, making CZE superior to gel electrophoresis and HPLC. Examples of therapeutic proteins that have been analyzed by CZE are recombinant human deoxyribonuclease (rhDNase) [46] and recombinant human erythropoietin (rhEPO) [47]. Recombinant human DNase is a complex phosphoglycoprotein that is used in the treatment of cystic fibrosis. Its molecular mass is about 34 000 Da and it contains two calcium binding sites. In the study by Felten and co-workers it was found that CZE of rhDNase using a polyvinyl alcohol coated capillary


16

and a background electrolyte of 200 mM 6-amino-n-caproic acid (EACA) at pH 4.8 and increasing amounts of calcium ions resulted in good separations [46]. In the study of the rhDNase it was also found that treatment of the analyte with neuraminidase and alkaline phosphatase resulted in significantly different electropherograms. The rhEPO was analyzed using a buffer containing 1,4-diaminobutane, which dynamically coats the capillary. Even though excellent separation of rhEPO glycoforms was achieved, the capillary was not useable after only a few injections, which was explained by non-specific adsorption to the capillary wall [47]. In the same study a capillary coated with dimethyl polysiloxane (DB-1) was evaluated for the separation of glycoforms of rhEPO using a 10 mM acetate buffer (pH 5.7) containing 0.5% (w/v) of HPMC as background electrolyte. In Fig. 5 electropherograms of a standard sample and a pharmaceutical preparation are shown. Even though the electropherogram from the pharmaceutical preparation has more peaks, the peaks in the 35-40 min region originate from the rhEPO glycoforms. This illustrates the potential of CZE to separate glycoforms of a glycosylated protein.

a b

Migration Time (min)

a ba ba b

Migration Time (min) Fig. 5 Electropherogram of an rhEPO standard (a) and a pharmaceutical preparation (b). The numbers 1-9 indicate the different glycoforms of the rhEPO standard. The RSD (%) of the migration times for the individual peaks was typically less than 2% (except for peak 9 which sometimes was not integrated). From [47]. In another study the ability of CZE to provide quantitative information on the separate peaks was evaluated [48]. This was done by analyzing a series


17

of concentrations from 0.2-5.0 mg/ml of rhEPO, and it was found that after normalization the peak areas can be used for quantitative analysis over the range investigated. These findings were considered very interesting, since gel isoelectric focusing, which was also used in their study, only provides semiquantitative data. In Fig. 6 separations of rhEPO by CZE and gel IEF are shown. Identification of each peak was performed through spiking with known components.

a ba b

Fig. 6 CZE (a) and IEF (b) of rhEPO, respectively. The numbers 1-6 indicate different isoforms separated. From

[48].

Size exclusion chromatography has also been used for the analysis of glycosylated EPO. In a study by Kendrick et al. [49] EPO from Chinese hamster ovary was analyzed by SEC and as can be seen in Fig. 7 no separation between the different glycoforms could be obtained, which illustrates the superior resolving power of CZE over SEC. However, in their study the aim was to distinguish between various PEGylated EPO’s, which was achieved (data not shown).


18

Fig. 7 SEC of EPO from Chinese hamster ovary. Dashed lines represent the part of the fraction that was subjected to further analysis. From [49]. CZE has also been employed for the characterization of insulin, e.g., for purity check [50], quantitation [15, 16], and for the monitoring of the formation of desamido-A21-insulin and desamido-B3-insulin [51]. In Fig. 8 an electropherogram of insulin and the desamido products is shown.

Fig. 8 CZE of human insulin and degradation products. From [51].


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Micellar electrokinetic chromatography (MEKC) MEKC is a mode of CE where a suitable detergent (e.g. sodium dodecyl sulfate, SDS) is added to the BGE, facilitating the separation of analytes as a result of both electrophoretic mobility (due to charge) and chromatography (due to partitioning of the analyte into micelles). Neutral molecules, which have no electrophoretic mobility, partition differently into the micelles according to their hydrophobicity. MEKC is applicable to a wide variety of compounds and sample matrices. Moreover, MEKC is one of the most robust variants of CE. Quite recently, MEKC was applied in the analysis of antithrombin III (AT III) variants [52]. AT III is a single-chain plasma glycoprotein that plays an important role in the regulation of blood clotting and it consists of two isoforms, i.e. the α- and the β-form, where the former is dominant with about 90% and the latter with about 10% in plasma. Patients with low levels of AT III are at a great risk of thrombosis, why AT III is of high clinical importance. In the paper by Dönges and co-workers [52] MEKC with cyclodextrin added to the background electrolyte was successful in the separation of AT III, while when cyclodextrin was not present no proper separation was achieved. As is evident from Fig. 9 baseline separation of the two isoforms of AT III was achieved with cyclodextrin present.

Fig. 9 MEKC of a mixture of α- and β-AT III. Buffer of 60 mM sodium borate, 40 mM SDS, 20 mM β-cyclodextrin, 1 mM diaminopentane, pH 9. Capillary 72 cm x 50 µm, detection at 200 nm, injection 0.5 s vacuum 169 mbar, potential applied 20 kV. From [52].


20

Capillary isoelectric focusing (CIEF) CIEF is a mode of CE where the capillary is filled with ampholytes. After the sample is injected, the analytes are first focused and then the focused zones are moved past the detector, for example by application of pressure. The proteins are separated on the basis of their isoelectric point (pI) in a pH gradient produced by the carrier ampholytes when an electrical potential is applied. CIEF is generally successful for proteins up to about 150 000 Da, which exhibit good solubility in aqueous buffer systems but may be unsatisfactory for larger or hydrophobic proteins. CIEF has the potential to be an effective tool for characterizing glycoforms based on differences in sialic acid content. The use of CIEF for the analysis of rhEPO [53] has been demonstrated. It was found that adding carrier ampholytes with pH 2.5-10 enabled the best resolution of rhEPO glycoforms. In the same publication CZE and CIEF were compared and it was concluded that the two modes of CE gave equally good separations and the general analysis time was shorter for CIEF but the reproducibility in terms of peak area of glycoforms was better in CZE. Tang et al. [54] developed a two-step CIEF method for the routine analysis of recombinant immunoglobulins (rIgGs). The proteins are first focused and then mobilized past the detector. It was found that this two-step CIEF method provided good resolution and reproducibility. CIEF was also successfully applied in the analysis of glycoforms of recombinant human tissue-type plasminogen activator (rt-PA), which is used in the treatment of myocardial infarction [55]. The molecular weight of rt-PA is about 59 000 Da and contains 527 amino acids and has three N-glycosylation sites, i.e. 117, 184 and 448. There are two main forms of rt-PA called type I and type II, where all glycosylation sites are occupied in type I while type II is only occupied in two positions, i.e. 117 and 448. CIEF was performed in capillaries with neutral coating and a diameter of 50 µm and a total length of 27 cm (effective length 20 cm). In Fig. 10 CIEF of rt-PA is illustrated. As can be seen, at least ten isoforms of rt-PA could be separated and detected. The baseline shift is caused by a change in background absorption during the analysis.


21

Fig. 10 CIEF of rt-PA using a 50/50 mix of pH 5-8 and pH 3-10 ampholytes. From [55].

Capillary gel electrophoresis (CGE) CGE is performed in either an immobilized polymerized matrix (a chemical gel) within the capillary (e.g. polyacrylamide) or in a free-flowing sieving matrix (physical gel). Such gels are needed in the analysis of analytes like DNA or SDS-protein complexes, where the charge-to-mass ratio is independent of size. Under these conditions, the separation is mainly due to the difference in size. In CGE the proteins are usually denatured before analysis by boiling the protein in the presence of β-mercaptoethanol, which breaks disulfide bonds, causing the protein to unfold. The sample is then mixed with sodium dodecyl sulphate (SDS) that binds to the protein. The presence of SDS leads to a uniform mass-to-charge ratio since it binds to proteins at a ratio of about 1.4 g/g protein [56]. For most proteins with a mass higher than about 10 000 Da it is true that the mass-to-charge ratio is uniform. However, for smaller proteins both phosphorylation and sulfonation may decrease the mass-to-charge ratio. For larger proteins, the separation of entities is thus purely based on difference in size, since the SDS/protein complexes have to migrate through the gel that fills the capillary. However, if the protein is glycosylated the mass-to-charge ratio is higher, since glycans do not bind SDS. Consequently, the migration time of such compounds is shorter than what otherwise would be the case [57]. In a study by Wu and Regnier [58] proteins with relative molecular masses of 14 000 to 205 000 were separated using non-crosslinked linear polyacrylamide gels in both coated and uncoated capillaries. In this investigation it was found that the viscosity of the acrylamide solution was a major factor affecting column stability. When


22

the viscosity is above 100 cP, electro-osmotically driven displacement of the gels is insignificant. CGE has been used for purity check of various proteins. For example, CGE has been successfully employed in the analysis of recombinant bovine somatotropin (rbSt), which enhances milk production in cows and has a molecular weight of about 21 000 [59]. Tsuji subjected production batches of rbSt to CGE and detected fragments, monomers, dimers and trimers of the protein. In Fig. 11 an electrophero-gram from this study is given.

Fig. 11 CGE of rbSt, where A is a fragment, B is the monomer, C is the dimer and D is the trimer of the protein.

From [59].

Tsuji also compared CGE with high performance size exclusion chromatography (HPSEC) and found that SDS gel-filled CE may be a good alternative to HPSEC for the analysis of recombinant proteins. A protein standard mixture containing proteins with a molecular weight from 14 000 to 97 000 was injected in the CGE capillary. After plotting the molecular mass and migration time on a logarithmic (log-log) scale a linear curve with a correlation factor of 0.998 was achieved. The relative standard deviation of molecular mass determinations was about 2-3%, and the CGE also proved to be highly reproducible with rather constant peak migration times even after 140 injections.


23

1.2.4 Mass spectrometry Over the last few decades biological and biomedical mass spectrometry has developed to the mature technique that it is today. The use of mass spectrometry in biomedical analysis was recently reviewed by Jonson [60] and Mano and Goto [61]. The development of soft ionization methods, such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI), has contributed to this since they can produce gas phase ions of large, polar, and thermally labile biomolecules, such as peptides and proteins, nucleic acids and others, and macromolecules larger than 100 kDa can easily be observed. The innovations of these ionization methods have led to a remarkable progress in mass spectrometric technology and in biochemistry, biotechnology and molecular biology research. Additionally, mass spectrometry is a powerful and effective technology employed in drug discovery and development. It can be used for studies on structural determination, drug metabolism, including pharmacokinetics and toxicokinetics, and de novo drug discovery by applying post-genomic approaches [61]. During the last about fifteen years there has been a steady increase in the yearly number of publications where ESI and MALDI are involved in the analysis within the biological and biomedical mass spectrometry. In 1987 the number of published papers involving ESI and MALDI was well under a hundred but had increased to a total of over 1500 published in 2001. Commonly used mass analyzers are the quadrupole mass spectrometers, the ion trap mass spectrometers, and the time-of-flight mass spectrometers [61]. In the quadrupole mass filter, ions produced in the ion source move with a vibration through four perfectly aligned electronically conducting cylindrical rods. The ion-trap mass analyzer also relies on ion separation in the electric field using a quadrupole. There is, however, a difference in the shape of the electrode and scanning mechanism. Ions introduced in the mass analyzer are trapped with a vibration in the space made between end-cap electrodes and ring electrodes. The time-of-flight detector measures the flight time of ions to the ion detector. Heavier ions travel at a lower velocity than lighter ions. This means that the TOF mass analyzer, in principle, has no mass range limit. However, the resolution is limited by the flight-time error, and the difference in the pulsed action and the energy aberration decrease the resolution in a TOF mass analyzer. One way to improve the resolution is to apply a mass reflectron, which


24

compensates for the initial energy and focuses ions having the same m/z value. Ions reaching the reflector are turned in the opposite direction by the electric field, and continue flying towards the detector. Electrospray ionization (ESI) ESI was first reported in 1968 when Dole et al. reported on the formation of gas phase ions by an electrospray process [62], and when Yamashita and Fenn demonstrated ion production and mass analysis using an ESI ion source coupled with a mass analyzer [61, 63, 64]. When an aqueous solution with uniform electrical density flows through a thin capillary, a split of charge is generated by adding a high voltage of several kV to the tip of the capillary, as illustrated in Fig. 12.

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proton affinity in the target molecule and that of the acidic component of the salt added to the mobile phase. An advantage of the ESI process is that it directly produces both single and multiple charged ions, which is useful for the mass analysis of macromolecules, such as peptides and proteins (because all mass analyzers separate ions according to their m/z ratio). This multiple charging can display very large molecules as an m/z ratio on a mass spectrometer with a relatively small m/z range, which allows an accurate measurement of the molecular weight.

The connection between an HPLC-system and an ESI-MS-instrument is performed by an interface. It is believed that the mass spectrometer acts as a concentration sensitive detector, i.e. the response should be


25

independent of the flow-rate. The current pneumatically assisted ESI interfaces are optimized for flow rates between 50 and 200 µl/min, and although it is claimed that MS instruments can handle up to 1 ml/min such high flow rates are usually not encountered in practice [65]. It is quite common that a splitter is used in order to decrease the flow from the LC to the MS, where one part of the flow passes for example a UV-detector and the rest goes to the MS. The connection of a CE-system to an ESI-MS-instrument is also quite straightforward. Like for HPLC-ESI-MS a pneumatically assisted nebulizer is employed. The capillary goes through a metallic tube through which a make-up liquid flows at a rate of a few microliters per minute. This make-up liquid is used to ionize the analytes coming from the capillary and is commonly prepared from formic acid or acetic acid with a concentration in the millimolar range. Since the flow from the CE is so small, it does not have to be split before entering the MS. However, due to dilution the sheath flow has a negative effect on the sensitivity.

The combination ESI-MS is also common in the analysis of proteins. For example, sonic hedgehog protein fused to an immunoglobulin Fc domain (Shh-Fc) is a therapeutically interesting protein that may be useful in the treatment of neurodegenerative disease and injury [66]. The engineering and expression of human Shh in Pichia pastoris was evaluated by Shapiro et al. [66] using ESI-MS to determine the molecular masses of the expressed proteins. As can be seen from the deconvoluted mass spectra in Fig. 13 analysis of the samples by ESI-MS enabled the authors to distinguish between several different forms of the proteins expressed. A deconvoluted mass spectrum is constructed from a normal mass spectrum where the ions originating from the same analyte are used in a mathematical equation to find the most probable molecular mass of the analyte.


26

Fig. 13 ESI-MS of Shh, with the native (45664), with a hexose adduct (45824), and a mixture of the unmodified and containing oxidized methione residues (45554). The deconvoluted spectra are shown. From [66]. Matrix-assisted laser desorption and ionization (MALDI) MALDI is, like ESI, a soft ionization method, and can be used for both polar and non-polar compounds [61]. In Fig. 14 the principle of MALDI is illustrated. The analytes are mixed with a matrix solution, which is an ultraviolet absorbing compound, and the mixture then co-crystallizes on the target plate.

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Fig. 14 Matrix-assisted laser desorption ionization source.

A pulsed laser beam irradiates crystals on the target plate, which causes the accumulation of high-density energy within the crystals. The concentration of energy causes the analyte and matrix molecules to vaporize, i.e. desorb, and results in proton transfer between the matrix and the analytes. Ideally, the target analyte exists as a homogeneous solid surrounded by the matrix


27

molecules, constituting a hydrogen bonding network, and this may also guard unstable analytes from energy pulsed laser power. MALDI produces mainly singly charged ions, and results in larger m/z ratios than those produced by ESI, which mainly produces multiply charged ions. Additionally, MALDI operates with pulsed lasers and is normally coupled to time-of-flight (TOF) mass detectors, which have no theoretical upper limit to the m/z ratio. The huge advantage with singly charged ions has made MALDI a routine method in the analysis of proteins and digests thereof. Nowadays, MALDI-TOF is the most common and primary analytical technique for peptides and proteins in proteomics research. There are many examples of off-line separation-MALDI-MS. After separation, fractions are collected and placed on the MALDI target for further analysis. Though this is not a difficult procedure it can be quite laborious and it is desired to minimize such tedious work. Therefore, attempts are made to perform “at-line” separation MALDI-MS. This is done by collection of entire chromatograms (or electropherograms) onto MALDI-targets. Quite recently, a publication describing CE-MALDI-MS was presented [67]. A vacuum deposition interface was used to get the analytes from the CE into the MALDI-instrument. The outlets of an array of capillaries (8 capillaries) were in contact with a moving polyester tape at vacuum, thus creating electropherograms on this tape. The tape can be analyzed either on-line or off-line. In the off-line mode the tape is transferred to an ordinary MALDI-target. Recombinant human erythropoietin (rhEPO) is a glycoprotein hormone that is used for the treatment of anaemia associated with chronic renal failure. The EPO consists of 165 amino acids with two disulfide bonds and about 40 w/w% carbohydrates, which contribute to the heterogeneity of the protein [68]. Recombinant human erythropoietin expressed in Chinese hamster ovary (CHO) contains four potential sites of glycosylation, i.e. one O-linked, serine (Ser) 126, and three N-linked, aspargine (Asn) 24, Asn 38, and Asn 83, respectively. In a study by Zhou et al. several analytical techniques were combined in order to characterize the intact as well as digests of rhEPO [68]. MALDI-TOF MS was employed to determine the molecular mass of the intact rhEPO. From Fig. 15 it is realized that the molecular weight of the molecule is 28 707 Da. This value is much lower than the 34 000 estimated by SDS-PAGE. However, the latter is known to give erroneous results for glycoproteins. The molecular weight of rhEPO as calculated from its amino acid sequence is 18 396, and the 10 311 Da


28

difference can be attributed to the carbohydrates attached to the protein [68]. Recombinant human erythropoietin is a protein that increases the red blood cell mass and it is consequently one of the most common doping substances in sports [69]. Often, the detection of this substance is accomplished by assays, e.g., bioassay or immunoassay. These methods are, however, insensitive but it has been suggested that HPLC-ESI-MS may provide the sensitivity necessary to reliably detect this substance in samples from athletes. The problem with EPO and its use in endurance sports as a performance enhancer stimulated Caldini et al. to develop a method to characterize rhEPO from three different manufacturers [70]. The substances were epoetin alfa, epoetin beta and darbepoetin alfa, which after separation by gel electrophoresis were subjected to analysis by MALDI-TOF-MS. Clearly, as is illustrated in Fig. 16, this approach enabled the differentiation between the three different rhEPO’s supplied by the various manufacturers and can consequently provide evidence that illegal doping has occurred.

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Fig. 15 MALDI-TOF-MS of rhEPO. The “Ref” represents the molecular ion. [mMnH]n+: multiply charged (n=1, singly charged; n=2, doubly charged, etc.) ion of oligomer (m=1, monomer; m=2, dimer, etc.). From [68].


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Fig. 16 MALDI-TOF-MS of epoetin alfa (A), darbepoetin alfa (B), and epoetin beta (C). In the lower trace the molar ratio B:C was 1:1. From [70].

Concluding remarks The advantage with ESI-MS is that in combination with suitable mass analyzers, e.g., quadrupoles, ion-traps or time-of-flight detectors, it provides high mass accuracy for proteins and the possibility to measure large proteins due to the multiple charging. Observing multiple peaks for the same peptide or protein allows the determination of molecular weight with high accuracy. With ESI it is also possible to perform MS/MS giving structural information by product ions generated by controlled fragmentation. MS/MS is suitable to establish the amino acid sequence and specific chemical modifications from the product ion pattern. However, even though the ESI-MS is such a versatile set-up it cannot easily be applied in the analysis of hydrophobic proteins. This is explained by the fact that detergents and/or salts frequently have to be used to get these proteins into solution. These compounds have a negative effect on the analysis through interference, which also has a detrimental influence on the sensitivity that can be obtained, that is, the purer the sample the higher the sensitivity. MALDI-TOF-MS, on the other hand, is less sensitive to contaminants (such as detergents, salts etc.). In addition, the sample preparation is fast and the sensitivity is rather high. For peptides levels of about low femtomole or even attomole are needed, while for proteins low femtomole


30

levels are required to achieve good signals. The mass accuracy that can be achieved with MALDI-MS is from ±5 ppm to 0.05% depending on in which mode the instrument is run, but the matrix material also has an influence on the result [71]. One potential problem with MALDI-MS is that the laser can induce degradation of the sample. The quadrupole-time-of-flight mass spectrometer (QTOF) is considered to be a powerful, robust and versatile set-up. Originally, it was used only for peptide analysis, but today it is applied for the analysis of such different sample types as biological samples using nanospray as well as common pharmaceutical samples for which higher flow-rate LC is used. The QTOF is characterized by a high mass accuracy and sensitivity. Also, its high mass resolution (m/∆m of about 10 000, where ∆m is the full peak width at half height), is very useful in the analysis of peptides and proteins, explaining the increasing popularity of this type of instrument in biological research [72]. 1.3 Technological aspects of the formulation of therapeutic proteins The administration of therapeutic peptides and proteins has been a challenge ever since the invention of the smallpox vaccine by Edward Jenner in 1796, which was later followed by the pioneering work of Louis Pasteur who developed vaccines against chicken cholera, anthrax and rabies. In a much later stage, this was followed by the introduction of insulin in the 1920’s and then by the introduction of thyroid hormone and Factor VIII. The increasing knowledge about the function of peptides and proteins can potentially lead to new tools in the struggle against cancer and other severe disorders. For example, interferons have found increasing clinical applications during the last 20 years [73]. Even though there has been a tremendous development in the field of protein chemistry and (bio)production, this has not yet been matched by a parallel development in the discipline of formulation and delivery of such drugs. The successful delivery of these drugs to patients depends on the solving of specific problems. Examples of such problems are 1) poor stability of the drug (storage in refrigerator or freezer is often necessary), 2) poor biovailability, 3) patient unfriendly dosage forms, usually the parenteral route, which might lead to non-compliance.


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1.3.1 Stability of peptides and proteins Due to biotechnological production processes, therapeutic proteins are generally obtained in an aqueous solution. When formulating these protein solutions, e.g., for parenteral delivery, stabilization of the protein is paramount and excipients that stabilize the protein and/or inhibit enzymatic breakdown have to be chosen. Also, even though peptides and proteins can be obtained with very high purities using the current biotechnological techniques, this does not guarantee improved stability. This is explained by the fact that the compound is no longer in its natural environment that usually contains carbohydrates, lipids and other proteins that can help stabilizing the protein. The stability of peptides and proteins in solution can also be affected by the buffer that is chosen. Buffers affect the stability of a protein in solution both by the ionic strength and the pH of the solution. Examples of buffers that have been used for this purpose are phosphate, citrate, maleate and succinate buffers. Even if the drug is in solution at the start of its shelf life, the challenge is to maintain the drug in solution. Peptides and proteins might aggregate during storage in solution, which can lead to precipitation of the drug, and degradation might also be caused by hydrolysis, deamidation, and denaturation, and also a number of other reactions. In Table III some common chemical and physical degradation mechanisms for proteins are given [24].

Table III. Some common causes of protein degradation.

Chemical degradation Physical degradation

Hydrolysis Denaturation

Deamidation Aggregation

Oxidation Adsorption

Disulfide exchange

β-elimination

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The solubility of proteins is pH-dependent, i.e. the solubility is lowest around the pI of the protein, but increases as the pH is changed away from the pI. However, at very low or high pH rapid H+ or OH- catalyzed degradation can occur. Temperature also influences the solubility, i.e. the solubility increases with the temperature, but above a certain threshold the


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protein may unfold leading to decreased solubility or even precipitation [24]. Since proteins are large molecules with many functional groups, they are susceptible to both physical and chemical degradation. This explains why proteins in solution are generally not stable for very long and commonly have to be stored at low temperature, e.g., in a refrigerator or even in a freezer [74]. The tertiary structure of proteins is vital to their function. This tertiary structure can be affected by the degradation mechanisms mentioned in Table III, which ultimately render a non-functional protein. Contrary to proteins, peptides do not posses a tertiary structure and are generally more stable. Many proteins can be stabilized in solution by using certain excipients. The mechanisms of stabilization by these excipients have been described by the so-called preferential interaction theory. This theory claims that the protein prefers to interact with water and the excipient is excluded from the domain of the protein and proportionally more water molecules are found at the surface of the protein than in the bulk. Preferential exclusion of an excipient is therefore usually associated with an increase in the surface tension of water. Examples of compounds that can be used as stabilizers of proteins in solution are amino acids, phosphate and sulfate, glycerol, erythritol and sorbitol. A more thorough discussion on this is found in previously published material [75-82]. Drying of peptides and proteins If the proteins could be stored in their dry form, it would be possible to achieve long-term stability, perhaps even at room temperature. An additional advantage of having the peptide/protein in a dry form instead of the dissolved state is that this would increase the versatility to develop various dosage forms. For example, the dry material can be processed into non-parenteral dosage forms, e.g., into tablets for oral delivery, or the dried powder can be used in a formulation for inhalation. Spray-drying and freeze-drying from a solution are the two most common methods to get proteins in the dry state. Stabilization of proteins through freeze-drying is frequently reported in the literature [83-86]. The importance of using excipients to stabilize proteins during drying is motivated by the fact that the drying process itself is rather harsh to proteins. During spray-drying, as solvent is removed, protein molecules are driven towards each other, which might lead to aggregation and there is also a mechanical stress


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exerted on the protein molecules during the vaporization phase. During freeze-drying aggregation may occur during the freezing phase due to the formation of ice crystals, i.e. freeze concentration. The formation of ice crystals can also cause degradation of the protein molecules due to the action of mechanical forces. Also, as solvent is removed the concentrations of other solutes increase that may also give rise to reactions between the protein and the solutes. A literature review reveals that especially sugars are the most successful excipients to stabilize proteins in the dry state. Sucrose and trehalose appear to be the sugars most often used [74]. A solution containing both protein and sugar properly dried yields a product where the protein is enclosed in a so-called sugar glass. It has been found that sugar glasses can provide long-term stability of the incorporated protein. When solutions of proteins with carbohydrates are freeze-dried the solution is first rapidly frozen in, e.g., liquid nitrogen. The use of liquid nitrogen ensures a very fast freezing of the solution. In the frozen state it is essential that the sample temperature is below the glass transition temperature of the maximally freeze concentrated fraction (Tg’). Above the Tg’ the freeze concentrated fraction is in its rubbery state and is therefore vulnerable to any reactions occurring. In addition, above the Tg’ the sugar may crystallize [87] by which no stabilization of the protein is obtained (see below). Moreover, if freeze-drying is performed below the Tg’ a porous cake will be achieved. A porous cake is often preferred because it is easy to reconstitute. On the other hand, during freeze-drying the sample temperature should be as high as possible to have a process of acceptable rate. Therefore, a high Tg’ is preferred. The Tg’ depends on the type of sugar used. The final dry product should also posses a glass transition temperature (Tg) that is as high as possible. The Tg of the end product depends on the type of sugar used and the water content [88-90]. Since water acts as a plasticizer and dramatically decreases the Tg it is desirable to keep the product as dry as possible. Also, if the product has a sufficiently high Tg it is less sensitive to moisture and it can be exposed to higher relative humidities at ambient temperature without turning into its rubbery state. A transition into the rubbery state would be disastrous to the protein since this leads to loss of protection. The protein would no longer be incorporated in a glass and thus be susceptible to all the reactions that it was meant to be protected from. Additionally, in the rubbery state the sugars may crystallize, which would lead to phase separation and thus the


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water replacement (see below) is no longer effective. During phase separation the proteins will also no longer be separated from each other and consequently be at risk of aggregation and further degradation. Phase separation is in itself a rather violent event that may harm the incorporated protein mechanically. In Fig. 17 an example of a state diagram illustrating the solid/liquid coexistence boundaries and glass transition profile of a binary sugar/water system is shown.

Fig. 17 A state diagram illustrating the processes during freezing of a binary sugar/water-system.

The solution is in the amorphous glassy state below and to the right of the glass transition line. Ice is present depending on the temperature and freezing path followed. Above and to the left of the glass transition line the solution is in the liquid state, with or without ice depending on temperature. For example, at point A the solution is in its liquid state. During cooling of the system to a temperature slightly below its equilibrium freezing point (due to undercooling), nucleation and crystallization begin at point B and initiate the freeze concentration process removing water in its pure form as ice. As crystallization of ice progresses the increasing solute concentration further depresses the equilibrium freezing point of the unfrozen portion (UFP) in a manner that follows the liquidus curve (towards C). During very rapid cooling freeze concentration continues past the eutectic point (Te) into a non-equilibrium state, since sugars generally do not crystallize as easily as water. As a result, the solution becomes supersaturated. After passage of a certain critical, solute-


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dependent concentration and temperature the unfrozen liquid suddenly exhibits very restricted mobility and the physical state of the UFP changes from a viscoelastic liquid (or rubber) to a brittle, amorphous glass. This critical point is the glass transition of the maximally freeze concentrated fraction with an accompanying temperature denoted as the Tg’ (point D in Fig. 17). At this point the supersaturated solution shows solid properties due to reduced molecular motion by which no crystallization of either water or sugar can occur anymore. During freeze-drying, first water is removed from the frozen system by sublimation of ice. This process is referred to as primary drying. Thereafter, water evaporates from the maximally freeze concentrated fraction. This process is referred to as secondary drying. As can be seen from Fig. 17, the glass transition temperature of the material gradually increases during secondary drying due to the decreasing water content. If during primary drying the sample temperature (accidentally) exceeds the Tg’, the material again transforms into supersaturated liquid (or rubber) with a high molecular mobility and sooner or later crystallization of the sugar will occur. As a consequence no sugar glass will be formed. Stabilization mechanisms of sugar glasses Three major theories have been presented to explain the mechanism behind the enhanced stability of proteins by incorporation into a sugar glass, i.e. the vitrification theory, the water replacement theory and the bulking effect theory. According to the vitrification theory, stabilization of proteins during drying is achieved by enclosure of the protein in a glassy structure [85, 91, 92]. The thermal stability of lactase incorporated in a trehalose sugar glass was evaluated by Mazzobre et al. [92]. They found that the activity of lactase decreased rapidly upon heating if the trehalose was allowed to crystallize. This indicates that the preservation of the glassy state is very important. In a glass the viscosity is in the order of 1012 Pa·s [93] and it is this extreme viscosity that increases the protein stability by slowing down the movements of reactants, and the rate of conformational changes of the proteins is also diminished. This means that the higher the viscosity of the glass the better it works as a stabilizer. Both sucrose and trehalose glasses have a high viscosity and this explains why they are good stabilizers during freeze-drying of proteins [94, 95]. As a matter of fact, it has been found and claimed that the disaccharide trehalose is superior to other saccharides regarding the stabilization of proteins during freeze-


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drying [92, 96-100]. Mazzobre and Del Pilar Buera later also investigated the freeze-drying of β-galactosidase with trehalose, i.e. incorporation of β-galactosidase in a sugar glass, and evaluated the effect of crystallization of trehalose [85]. It was found that crystallization of trehalose was accelerated by higher amounts of moisture present. As predicted by the vitrification theory, crystallization of trehalose had a detrimental effect on the stability of the incorporated β-galactosidase. The water replacement theory was presented by Carpenter and Crowe in 1988 [101, 102] and states that protein stabilization during drying is achieved by the ability of carbohydrates to take the place of water around the protein during the drying process. Later, this was further investigated by Carpenter and Crowe who used Fourier transform infrared spectroscopy (FT-IR) to assess the interaction between carbohydrates and proteins [103]. Solutions of lysozyme and various carbohydrates, i.e. trehalose, lactose or inositol, were freeze-dried and the product was analyzed by FT-IR. It was found that the carbohydrates interacted with lysozyme in the dry product in a way similar to the water/protein interaction in solution. Some carbohydrates serve as water substitutes and thereby preserve the native structure of the protein [75]. The intra- and interprotein hydrogen bonding may in this way be prevented during dehydration [98]. Therefore, stabilization of proteins requires hydrogen bonding with an excipient during drying. During the drying process, as water is removed, the hydroxy groups of the excipient takes the place of the water, thereby achieving stabilization [75, 91, 104]. Stabilization of proteins by the bulking effect is explained by the “dilution” of the protein in the matrix due to the presence of large quantities of compounds other than the protein. This leads to a lower probability of protein aggregation or the occurrence of degradation reactions. Even though these theories may seem totally different, it does not mean that only one of them is correct. They may all be valid and explain (each a part of) the stabilization of proteins in solution as well as during/after drying. As already mentioned, it is desirable that the sugar glass possesses a Tg as high as possible, since this will enhance its stabilizing capacity. If the sample is exposed to a temperature higher than the Tg the glass will transform into its rubbery state, which would lead to the loss of vitrification and, in addition, crystallization may occur. Crystallization


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leads to phase separation and thus lost stabilization due to the loss of bulking effect and the water replacement theory is no longer valid. It is also known that the Tg is depressed by moisture [105], which further stresses the importance of a high Tg and low hygroscopicity. In a recent study it was demonstrated that the polysaccharide inulin is a sugar that satisfies both these demands. Hinrichs et al. [106] lyophilized alkaline phosphatase with glucose, trehalose and two inulin types with a degree of polymerization of 14.2 and 23, respectively. It was found that after storage of the lyophilized product for six days at 60 °C, only the alkaline phosphatase lyophilized with either of the inulins still demonstrated enzymatic activity. Moreover, it is necessary to employ non-reducing sugars since otherwise the Maillard reaction may be a problem. In this reaction the amine functions of the protein react with the reducing groups of the sugar, causing degradation of the protein. In a study by Schebor et al. [96] it was shown that browning and loss of enzymatic activity of invertase lyophilized with raffinose, lactose, or maltose and subsequently stored at 95 °C for 7 days correlated. Invertase that had been lyophilized with trehalose or sucrose, which are non-reducing sugars, did not show this behaviour, i.e. no browning occurred. However, the invertase lyophilized with sucrose lost enzymatic activity due to crystallization after storage at elevated temperature, which did not occur for the trehalose sample. In addition to these physical properties the stabilizing excipient must also be non-toxic and preferably described in the USP and Generally Recognized as Safe list (GRAS-list). 1.3.2 Drug delivery Delivery by injections Currently, most systemically acting peptides and proteins are delivered via the parenteral route. This can be ascribed to the fact that although many new therapeutic agents recently evolved from biotechnology, only minor attention has been paid to the development of stable formulations that can be used by the patient by more comfortable routes of administration. Another reason is that these drugs have to pass enormous barriers if delivered via other routes, e.g., enzymatic degradation and penetration barriers. There are a number of peptide/protein-based drugs on the market, insulin being the most familiar example. Other


38

examples are Calcitonin, Erythropoietin, Factor VIII, human growth hormone (somatotropin), and a number of interferons. A disadvantage of many peptides and proteins is their very short half-life in the body, commonly only a few minutes. This demands repeated injections during the day, which is inconvenient for the patient. Therefore, efforts are made to increase the half-life of such drugs. This can be accomplished by making controlled release formulations, where a single injection may assure adequate drug plasma levels up to one month or even longer. Examples of controlled release parenteral formulations are polymeric implants, oily injections or particulate systems. A suspension of γ-interferon in oil was reported to prolong the activity of interferon when administered by subcutaneous injection (s.c.) to beagles [24]. Particulate systems are exemplified by microspheres, microparticles, and microcapsules, which are larger than 1 µm and nanocapsules, nanospheres and nanoparticles [24], which are smaller than 1 µm. In general, these microparticulate systems are administered via subcutaneous (s.c.) or intramuscular (i.m.) injection. Various polymers have been described for use in slow release injectable products like implants or microspheres. Examples are polyglycerol esters of fatty acid (PGEFs), polylactic glycolic acid (PLGA), polylactic acid (PLA), gelatine hydrogels, and polyethylene glycol (PEG) [107-112]. Parenteral formulations can also be delivered by continuous infusion via i.m., i.v., or s.c. injection. Freeze-dried or spray-dried formulations of proteins, i.e. stabilized proteins, can also be delivered via the parenteral route, i.e. after reconstitution in a suitable solvent. Delivery via the ocular route An alternative to injections is delivery via the ocular route, i.e. administration via the conjunctival sac under the eye. Christie and Hanzal suggested this already in 1931 as a possible route of administration of insulin [113]. Since then, a number of other peptides and proteins have been investigated. The ocular route of delivery offers a few advantages, e.g., the absorption is relatively fast and the absorbed polypeptide bypass the portal circulation to the liver, thus avoiding the first-pass metabolism by the liver [24]. Even though it has been shown that the eye is a possible route of administration of drugs, there are some important drawbacks of this approach. For example, the bioavailability of proteins administered through the ocular route is low due to their large molecular size.


39

Additionally, the eye is very sensitive to foreign substances, and most people are instinctively very protective of their eyes and clinicians are concerned about local toxicity. Nonetheless, for the treatment of ocular disorders it is advantageous to deliver drugs immediately to the eye itself. Several types of dosage forms can be applied as delivery system for the ocular delivery of drugs. The most prescribed dosage form is the eye drop solution, which is easy to administer but has one important drawback; the drug solution is immediately diluted in the tear film as soon as it is instilled into the cul-de-sac and is drained away from the precorneal cavity by the constant tear flow. This tear flow is also more intense in an irritated eye than in a healthy eye. Therefore, quite concentrated drug solutions must be applied to achieve an adequate therapeutic effect. This procedure leads to massive and unpredictable doses of medication, which may also cause unwanted side effects. On the other hand, it has been found that the cell permeability is higher in an inflamed eye. Further reading on the subject of ocular drug delivery can be found in review articles by Zimmer and Kreuter who concentrated on the use of microspheres and nanoparticles [114], while Harris et al. concentrated on the problems with ocular delivery of peptides and proteins in general [115]. Delivery via the nasal route In addition to its primary function as a heat exchanger and an organ to filter particulates, the nose is a possible route for the delivery of drugs. In an adult human the total surface area of the nasal mucosa is approximately 160-180 cm2 and the rich vasculature of the nose makes absorption of drugs possible. Because of this, drugs delivered to the nose lead to a fast onset of action, almost comparable to that after intravenous injection. Delivery via the nose is also advantageous since it provides a patient friendly manner of drug delivery. In addition, drugs absorbed through the nasal mucosa bypass the hepatic circulation and thereby first-pass metabolism, which increases the half-life of the drug in the body. In Table IV a few commercially available peptides that are delivered via the nasal route are given.


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Table IV. Examples of peptide drugs that are administered via the nasal route by means of nasal spray.

Peptide Therapeutic indication

Lypressin nasal solution USP (Diapid®, Sandoz) Diabetes insipidus

Desmopressin (DDAVP®, Rhône-Poulenc Rorer),

(Stimat®, Armour/Ferring

Diabetes insipidus, Primary nocturnal enuresis

Oxytocin nasal solution USP (Syntocinon®, Sandoz)

(Stimate®, Armour/Ferring)

To assist initial postpartum milk ejection

Nafarelin (LHRH-agonist) (Synarel®, Syntex) Endometriosis

Adapted from Banga [116].

In general, the nasal route is suitable for systemic delivery of molecules with a molecular weight less than about 1000 Da. For these molecules, the nasal bioavailability may be sufficient to achieve therapeutic levels without the use of any adjuvants, i.e. permeability enhancers. With the use of adjuvants, this limit can be pushed up to at least 6000 Da and perhaps even higher [117]. A therapeutically relevant peptide drug that has been extensively studied is insulin. The nasal delivery of insulin in rabbits was studied by Callens and Remon [118], who freeze-dried dispersions of insulin with Carbopol 974 P and starch or maltodextrin. The dry material was then passed through a sieve to get particles smaller than 63 µm. After nasal delivery to rabbits it was found that the bioavailability was higher for the Carbopol 974 P/starch mixture than for the Carbopol 974 P/maltodextrin mixtures. In a later study Callens et al. evaluated the toxicological effects of a bioadhesive nasal powder containing Carbopol 974 P/starch on the rabbit nasal mucosa and slug mucosa, finding that the formulation was not irritating [119]. Later, the influence of multiple nasal administrations of bioadhesive powders on the insulin bioavailability was evaluated by Callens and co-workers [120]. After a single administration the bioavailability of insulin was found to be about 10%, but repeated administration led to decreasing bioavailability. This was explained to be an effect of the high viscosity of the bioadhesive powders, causing a physical barrier towards absorption and a strongly reduced mucociliary clearance. Calcitonin is a 32 amino-acid polypeptide hormone that is used in the treatment of bone diseases such as Paget’s disease, hypercalcemia and osteoporosis. The nasal route of delivery is one of the routes that have been studied and it has been found that the absorption is significantly higher


41

when promoters are employed [121]. In the previously mentioned study Calcitonin was delivered with and without 0.5% sodium tauro-24,25-dihydrofusidate (STDHF) and it was found that the bioavailability was up to 4.7 times higher with STDHF. In a review by Pontiroli current and emerging uses of nasal delivery of peptide hormones were highlighted [122]. In this review a number of peptide hormone formulations for nasal delivery are presented which are already on the market, e.g., desmopressin that is used in the treatment of diabetes insipidus, oxytocin that is used for labour induction and buserelin that is used for treatment of prostatic cancer. Common for these peptide hormones is that they are all rather small, less than 10 amino acids in size. This means that the bioavailability is quite high even without absorption enhancers [122]. Delivery via the pulmonary route Another non-invasive route of delivery is via the pulmonary route. In fact, this route of delivery is by no means new or revolutionary. The inhalation of various drugs is well established and can be traced back to the earliest civilizations. The most obvious example is, naturally, tobacco, which was brought back from America to Europe by Christopher Columbus in the 15th century, but had probably been used by American natives for thousands of years. Moreover, inhalation of sea mists, hot vapors and aerosols to ease airway obstruction were remedies used by Hippocrates, and the smoking of leaves of Atropa Belladonna to suppress coughing dates back about 4000 years [123]. Efficient delivery of effective amounts of drug to the lungs is, however, not as simple as it seems. First, the inhaled drug has to travel from the mouth and turn about 90 degrees in the oropharynx without depositing at the back of the throat. Then, there is the progressive branching and narrowing of the airways down the bronchial tree. In all, there are about 23 bifurcations from the mouth to the alveoli. In Fig. 18 an image of the lungs is given.


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Fig. 18 Image of the lungs demonstrating the branching and complexity of this organ.

Briefly, the human respiratory system can be divided in four regions, i.e. the upper respiratory tract (heats and moistens the air), the conduction zone (the first sixteen generations of branches beginning with the trachea and ending with the terminal bronchioles), the transitional zone (branch generation seventeen to nineteen) and finally the respiratory zone ending with the alveoli. This system presents large obstacles for an inhaled particle to find its way into the deep lung. Moreover, high humidity and mucociliary clearance mechanism further complicate matters and may inhibit the penetration of inhaled drug particles into the deep lung [124]. An important factor affecting the drug deposition in the respiratory tract is the “mass median aerodynamic diameter” (MMAD). This is a function of particle size, shape and density. Also important is the particle charge and patient specific characteristics, such as respiratory rate and the occurrence of, e.g., asthma, bronchitis or emphysema. The successful delivery of drugs to the lungs requires the MMAD being in the range 1-5 µm, which is commonly referred to as the “fine particle fraction” (FPF). If the lower regions of the lung (preferably the alveoli) have to be reached an even smaller size distribution is required (1 – 3 µm) [124]. If the particles are smaller than 1 µm significant fractions will be exhaled again during normal breathing, while if they are bigger than 6 µm they will not make it into the lung, but deposit at the back of the throat and eventually be swallowed [125]. Reaching the alveoli is important if systemic absorption is sought


43

since the permeability of the alveolar membrane for polypeptides and proteins is much larger than that of the airway membrane. On the other hand, for local effect reaching the alveoli is not necessary, since just delivering the drug to the airways may already cause a therapeutic effect. Systemic drug delivery via the pulmonary route has the advantage that the first-pass metabolism by the intestinal membrane and the liver is avoided. The lung is regarded as a promising route for the delivery of peptides and proteins, since it is less invasive and better accepted by patients than injections and though there is some degrading enzymatic activity it is low compared to the gastrointestinal tract. Moreover, the surface area of the alveoli is an impressive 43-102 m2, comparable to a tennis court. Additionally, the walls of the alveoli in the deep lung are very thin, typically 0.1-1 µm in thickness and relatively large tight junctions (diameter of about 5 nm) between the cells exist. Therefore it has been predicted that absorption of peptide and protein drugs with a molecular mass up to about 30 kDa should be fast from this organ [125]. As an example, γ-interferon (19 kDa) had 56% bioavailability [126]. Studies have shown that, without surfactant enhancers, the lungs provide substantially greater bioavailability for macromolecules than other routes of delivery [127]. For a drug to enter the blood stream from the lungs it must pass several barriers, i.e. the surfactant layer, the surface lining fluid, the epithelium, the interstitium and basement membrane and, finally, the vascular endothelium. It has been demonstrated that the absorption of large molecules in the lung is partially diffusion controlled. Inhaled molecules with a mass above about 40 kDa are slowly absorbed over many hours from the airspaces. After “absorption” they appear in higher concentrations in pulmonary lymph (relative to plasma) during absorption [127]. However, the absorption rate and bioavailability of these larger proteins are too low to be of therapeutic relevance. Pressurized metered dose inhalers (pMDI) have been used for many years to accomplish the delivery of drug containing (dry) particles to the lung. Unfortunately, these systems have some drawbacks, such as the difficult hand-lung coordination by the patient and the use of environmentally damaging propellants such as CFC’s. Nebulizers, on the other hand, are very efficient in creating mists of fine droplets from drugs in solution that in this manner can be delivered to the lung. Unfortunately, it may take over 30 minutes for a patient to inhale a dose from a nebulizer and proteins may be damaged by the aerosolization process. Therefore, new devices, such as the dry powder


44

inhalers (DPI), have been developed. The advantage with these inhalers is that they are breath-actuated and so coordination problems such as synchronizing dose discharge with inhalation are overcome. Moreover, they can be applied for formulations containing the (stabilized) dry protein. This is, however, a new development and still in the experimental phase. It has recently been shown that with the dry powder inhalers an improved deposition to the lung can be achieved [128, 129]. One proteinous drug that has been extensively investigated regarding pulmonary delivery is insulin. Patton et al. have reviewed the potential of inhalable insulin [130]. Another drug that is routinely delivered via the pulmonary route is recombinant human deoxyribonuclease that is used in the treatment of cystic fibrosis. However, this protein exerts its effect locally and not systemically [123]. Some review articles on this subject that give more information on the state of the art have recently been published [123, 124, 131, 132]. Delivery via the oral route The ultimate challenge is to be able to deliver therapeutic peptides and proteins via the oral route. However, the obstacles that have to be overcome are at a first glance almost insurmountable. Peptides and proteins are chemically and physically unstable. As the drug enters the stomach it is exposed to the harsh, acidic environment. One possibility to protect the drug from the acidic environment is to provide the tablet with an enteric coating. The principle is that the coating is stable at acidic pH but starts dissolving as the pH increases above a certain threshold, e.g., pH 6, and the drug is consequently released in the intestinal lumene. Over the whole gastrointestinal tract the peptides/proteins are exposed to digestive enzymes, e.g., proteases and peptidases. If the drug survives this, it must penetrate through the intestinal membranes and overcome first pass metabolism and the rapid clearance of proteins in the body [133]. Because of the multitude and variety of proteases in the intestinal tract, the enzymatic barrier is considered the most important barrier limiting the bioavailability of protein drugs. A further discussion on the relation of peptidases and oral peptide drug delivery can be found in a review article by Bai and Amidon [134]. The intestinal epithelial barrier separates the internal tissues from the external environment. Its main functions are protection from the outside environment, terminal digestion and selective absorption, and secretion of water, electrolytes etc. As for other epithelia, it is a natural barrier for unwanted substances [135]. The intestinal epithelium


45

is made up of a single cell layer, and these cells are connected to their neighbors by a junctional complex creating pores with a size of about 3-9 Å [133]. Underlying the epithelium is the lamina propria, which in turn is supported by the muscularis mucosa. These three together are referred to as the intestinal mucosa. In Fig. 19 an illustration of the intestinal wall is given.

Fig. 19 An illustration of the intestinal wall. From [136].

Because the intestinal tract is lined with phospholipids, hydrophilic proteins cannot easily diffuse through it (by passive paracellular diffusion), and the size of protein molecules prevents them from entering the pores. Nevertheless, passive diffusion across the intestinal membranes has been demonstrated not only for small proteins such as insulin (5.8 kDa) but also relatively large proteins such as bovine serum albumin (66 kDa) [133]. This passive diffusion of macromolecules, such as proteins, is likely to occur through the transcellular pathway [137]. The paracellular pathway is not of major importance, since the pore size is not large enough. The small intestine epithelial mucosa is structured in such a way that it constitutes a barrier to the permeation of macromolecules. However, endocytotic uptake provides ways to circumvent this barrier through a form of active transport called receptor-mediated endocytosis (RME). RME allows the absorptive cells of the intestinal epithelium to select and transport specific molecules while excluding undesirable or potentially harmful ones, e.g., bacterial enterotoxins and endotoxins. Thus, depending


46

on their molecular structure, endogenous and foreign macromolecules may interact with the intestinal epithelial mucosa receptors and subsequently undergo internalization [138]. Indeed, some peptides and proteins are known to enter intestinal mucosal cells through endocytosis. In Table V some proteins that are known to enter the intestinal mucosa through endocytosis are given. It has also been found that lactoferrin (an iron-binding protein in milk) enters the systemic circulation via endocytosis. This was shown by Harada et al. [139] in an investigation where neonatal piglets were fed with bovine lactoferrin, and the transport of lactoferrin was evaluated by immunohistochemical methods.

Table V. Proteins and large peptides transported across the intestinal mucosa.

Protein or peptide Species used in study

Insulin Human (adult), Rat

Horseradish peroxidase Rat

Egg albumin Human (infant)

Trypsin and chymotrypsin Human

Chymotrypsin Rat

Γ-globulin Neonatal pig, neonatal mouse

Native ferritin Rat

Immunoglobulin G Rat

Epidermal growth factor Rat

Nerve growth factor Rat

Reproduced from [138].

Most mammalian cells, including those of the intestinal tract, have some capacity to endocytose macromolecules [140]. For example, Peyer’s patches (PP), discovered already in 1676 by Peyer, belong to the gut-associated lymphoid tissue (GALT) and consist of aggregated lymphoid follicles. PPs are oval or rectangular and extend through the luminal epithelium, lamina propria, and lamina submucosa, and are only one follicle thick. The antigenic stimulation of the intestinal mucosa is correlated with the development of PP, and one patch can contain 5-250 lymphoid follicles. Transport across the epithelium in PP can occur by endocytosis, via the paracellular shunt, or passive diffusion. The endocytotic activity seems to be dependent on age and health state of the individual and it has also been found that several microorganisms can


47

enter the circulation via the PP [140]. Despite the problems that are associated with oral delivery of peptides and proteins, it is motivating to see the possibility of local therapy of such drugs when delivered to the intestinal system. Due to the fact that proteins are subjected to various digestive processes, the oral delivery of peptides and proteins is not commonly encountered. There are, however, a few examples of such drugs. One of the most investigated protein drugs for oral delivery is insulin. Ever since its initial discovery by Banting and Best in 1922, an oral dosage form of insulin has been the goal for a multitude of investigators [141]. For insulin a controlled release system was developed by Marschutz et al. [142], who claimed that mucoadhesive polymer-inhibitor conjugates might represent a promising excipient in delivery systems for oral (poly)peptide delivery. These conjugates were made of carboxymethyl-cellulose (CMC)-Bowman-Birk inhibitor and CMC-elastatinal and were homogenized with polycarbophil-cystein conjugate, insulin, and mannitol. It was found that in the formulation containing the inhibitor conjugate, insulin was more stable than in the formulation without the inhibitor when evaluated in an in vitro system. As an extra challenge for insulin, apart from all the common digestive enzymes, is the fact that there also exists a cytosolic insulin-degrading enzyme [143, 144], which makes the oral delivery of insulin even more difficult. The therapeutic potential for orally administered type 1 interferons has been reviewed by Beilharz [73]. In this review it can be found that orally administered interferons may be useful in the treatment of multiple sclerosis, Sjögren’s syndrome (dry mouth), and hepatitis B among others. Also, it has been found that when small amounts of interferon are administered orally the effect is almost always better than when high amounts are delivered [145]. This is explained by the assumption that low doses given orally mimic a natural host/defence process. Delivery via the colon and rectum The human rectum is the final 15-20 cm of the large intestine (colon). The rectal epithelium does not contain villi and its surface area is an unimpressive 200-400 cm2. It is rich in blood vessels, lymphatic vessels and microflora. Three types of haemorrhoidal veins permeate the mucous membrane, i.e. the inferial, middle and superior. The inferior and middle veins are directly connected to the systemic circulation, while the superior


48

is connected to the portal system. Drugs that are administered in the lower region will therefore bypass the hepatic first-pass metabolism to a large extent. Compared to the small intestine and stomach, the proteolytic activity in the rectum is very small, and is consequently a potential mucosal route for the delivery of peptides and proteins. In the presence of adjuvants a high systemic bioavailability may be possible. However, drug absorption may be erratic and may be interrupted by defacation [116]. The most investigated protein for rectal delivery is insulin, probably due to its therapeutic importance. Both gels and suppositories have been evaluated. However, the availability of insulin after rectal delivery is poor due to the mucosal epithelial cell layers that pose a serious barrier to all peptides and proteins. Therefore, some kind of absorption enhancers is frequently used. Barichello et al. [146] used insulin-loaded Pluronic® F-127 gels containing unsaturated fatty acids (oleic acid, eicosapentaenoic acid, and docosahexaenoic acid) in normal rats. They found that the availability of insulin was about 25% with the enhancer compared to only about 5% without, a result that was also found by Onuki et al. [147]. Cyclodextrins have also been evaluated as enhancers of rectal uptake of insulin. It was, for example, found by Watanabe et al. [148] that when cyclodextrins were used the uptake was significantly better than when no cyclodextrin was present in a study performed in rabbits. Van den Mooter and co-workers evaluated the in vitro release properties of methacrylated inulin hydrogels intended for delivery to the colon via the oral route using lysozyme and bovine serum albumin as model proteins [149]. They found that the food composition and the degree of substitution of inulin are important in controlling the extent and rate of release. It was also found that the presence of inulinase led to increased release, which indicates that the methacrylated inulin is indeed biodegradable. Interestingly, inulinase is only present in the colon and not in the other parts of the intestinal system. In a recent review by Chourasia and Jain [150] strategies to perform drug delivery to the colon via the oral route are addressed. There are several suggestions how to achieve colon drug delivery. Tablets containing the drug, e.g., a peptide or a protein, can be coated with polymers that are stable at acidic pH but dissolve once the pH increases to near neutral conditions such as those in the colon. This approach was investigated by Morishita et al. who coated insulin microspheres with Eudragit L100 and S100 [151]. The drug release from the microspheres was evaluated by an in vitro model, i.e. a rotating basket dissolution testing system using


49

phosphate buffers at pH 6 to 7.5. The microspheres coated with Eudragit L demonstrated faster dissolution than those coated with Eudragit S. In the in vivo model it was also found that insulin containing microspheres coated with Eudragit L100 provided the highest efficacy. Drugs can also be coated with polymers containing azocompounds. Such polymers are resistant to digestive enzymes, like those present in the stomach and the small intestine, but are digested by the bacteria present in the large intestine. Once dissolved, the drug can act either locally in the colon or even be available for absorption into the blood. This approach has been studied for a number of drugs, e.g., insulin and vasopressin [152]. It was found that administration of vasopressin and insulin, respectively, coated with azoaromatic polymer-coated capsules or pellets resulted in a desirable biologic effect, i.e. antidiuresis and reduced blood glucose levels, respectively. Absorption enhancement Delivery of peptides and proteins into the systemic circulation is in principle not feasible due to the epithelial and enzymatic barriers that have to be overcome. Peptide and protein drug delivery is consequently often combined with penetration enhancement and efforts to suppress enzymatic activity in, e.g., the digestive tract. The penetration enhancers increase the absorption by one or more of the following mechanisms: (1) altering rheologic properties of the mucus layer, (2) fluidizing the membrane lipid layer, (3) altering the intracellular tight junctions and (4) inhibiting enzymatic activity [133]. The use of penetration enhancers is, however, not without risk, since the increased absorption is not only for the peptide or protein for which it is intended, but for all compounds. Therefore, some unwanted toxic effects might occur when such substances are used in the formulation. It has also been found that the use of microparticulate delivery systems as drug/vaccine delivery carriers via mucosal routes give enhanced absorption and was recently reviewed by Hillery [153]. Protease inhibitors have also been investigated and have been found to increase the absorption of peptides and proteins in the mucosal membrane. Examples of protease inhibitors that have been investigated are bacitracin, aprotinin, soybean trypsin inhibitor, bestatin, and puromycin [154].


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1.4 Alkaline phosphatase In this introduction some of the analytical and technological aspects of pharmaceuticals based on peptides and proteins in general have been addressed. A protein that is thoroughly investigated in this thesis is alkaline phosphatase. Alkaline phosphatases (E.C. 3.1.3.1) are actually a group of cell-membrane-associated enzymes with hydrolase and transferase activity that act on a variety of phosphate substrates. They are structurally and functionally related, i.e. they have high pH optima, their activity is dependent on zinc and magnesium ions, and they have a dimeric structure and suggested monomer molecular masses of about 58 000 [155] and 65 000 Da [156, 157], respectively. The isoelectric point (pI) of alkaline phosphatase is about 4.5 [156]. In humans and other higher organisms there are four isoforms of this enzyme, the intestinal, germ cell, tissue non-specific (liver, bone, kidney) and the placental form. The placental form is more heat stable than the other three isoforms, which lose their enzymatic activity when treated at 56 °C for 30 min, while the placental form has been found to withstand a temperature of up to 70 °C for 30 min [158, 159]. The presence of alkaline phosphatase in human serum was demonstrated already in the 1920s [160] and later its clinical relevance was evaluated [35, 157, 161-165]. In 1997 a possible physiological function of the enzyme was presented by Poelstra et al. [166, 167], who proposed that alkaline phosphatase might play a significant role in the detoxification of endotoxins produced by Gram-negative bacteria. This is achieved by enzymatic removal of the phosphate groups present in the lipopolysaccharides produce by such bacteria. This important finding makes alkaline phosphatase an interesting candidate drug for the treatment of sepsis, which is a serious condition in which endotoxins play a significant role. 1.5 Scope of the thesis In the near future the impact of protein pharmaceuticals will strongly increase. Already today, the therapeutic effectiveness of such biopharmaceuticals is studied extensively and several are used in therapies. As any other drug, pharmaceutical proteins have to meet high standards regarding efficacy and safety. However, as biopharmaceuticals represent


51

complex molecular structures and samples, this puts new challenges towards analysis and formulation. In this thesis new analytical and formulation procedures for protein pharmaceutics are investigated using alkaline phosphatase, a potential therapeutic protein, as test compound. From the analytical perspective, emphasis is put on the potential role of CE in purity and stability analysis. Also the possibility to couple CE with MS for the characterization of intact proteins is considered. The formulation research is focused on the use of inulin sugar glasses to stabilize and formulate proteins. Chapter 1 gives an overview of the literature on analytical techniques and formulation aspects regarding therapeutic proteins. In chapter 2 the combination of several analytical techniques to monitor the progress of a purification process of placental alkaline phosphatase is investigated. The extent of purification using preparative ion-exchange chromatography and affinity chromatography is examined. In particular data obtained by CZE, CGE, MALDI-TOF-MS and enzymatic activity assay are evaluated and compared. In chapter 3 the potential of capillary electrophoresis to monitor the forced degradation of alkaline phosphatase is examined. The potential of CZE for the analysis of alkaline phosphatase that has been exposed to various stressful conditions is assessed by studying the separation and detection of several degradation products. In chapter 4 the question is raised and studied whether it is possible to characterize intact proteins by on-line coupled CE-MS while using non-volatile buffers as background electrolyte. The proteins investigated are insulin, myoglobin and bovine serum albumin, and phosphate and borate buffers are tested. In chapter 5-7 the formulation of proteins is considered. In previous studies it has been found that inulin has properties that make it a promising compound for stabilization of proteins as well as production of tablets. In this thesis these findings are combined in an effort to evaluate the possibility to make tablets of proteins incorporated in inulin sugar glasses. The influence of various buffer components on the glass transition temperature of amorphous inulin is evaluated in chapter 5. The possibility to stabilize alkaline phosphatase by freeze-drying from different protein/sugar solutions followed by compaction is evaluated in chapter 6. The two sugars trehalose and inulin are compared. In chapter 7 results from a proof of concept study regarding the delivery of freeze-dried and compacted alkaline phosphatase to the intestinal mucosa of a rat by oral administration are presented. Alkaline phosphatase incorporated in


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amorphous inulin is compacted to tablets with a diameter of 4 mm that subsequently are coated with an enteric coating.


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1.6 References 1. www.ICH.org, International conference on harmonisation of technical

requirements for registration of pharmaceuticals for human use. 2. Welling, G.W., R. van der Zee, and S. Welling-Wester, Column liquid

chromatography of integral membrane proteins. J Chromatogr, 1987. 418: p. 223-243.

3. Aguilar, M.I. and M.T. Hearn, High-resolution reversed-phase high-

performance liquid chromatography of peptides and proteins. Methods Enzymol, 1996. 270: p. 3-26.

4. Burton, W.G., K.D. Nugent, T.K. Slattery, B.R. Summers, and L.R.

Snyder, Separation of proteins by reversed-phase high-performance liquid chromatography. I. Optimizing the column. J Chromatogr, 1988. 443: p. 363-379.

5. Protein Liquid Chromatography. Journal of Chromatography Library,

ed. M. Kastner. Vol. 61. 1999, Amsterdam: Elsevier. 974. 6. Hearn, M.T.W. and B. Grego, High-performance liquid chromatography

of amino acids, peptides and proteins. J Chromatogr, 1981. 218: p. 497-507.

7. Moffatt, F., P. Senkans, and D. Ricketts, Approaches towards the

quantitative analysis of peptides and proteins by reversed-phase high-performance liquid chromatography in the absence of pure reference sample. J Chromatogr, 2000. 891: p. 235-242.

8. Issaq, H.J., T.P. Conrads, G.M. Janini, and T.D. Veenstra, Methods for

fractionation, separation and profiling of proteins and peptides. Electrophoresis, 2002. 23(17): p. 3048-3061.

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university of groningen analytical techniques and ...1000 da) the characterization of purity is...

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