Low Molecular Weight Excipients Research Articles

The effect of mAb and excipient cryoconcentration on long-term frozen storage stability – Part 1: Higher molecular weight species and subvisible particle formation

Cryoconcentration upon large-scale freezing of monoclonal antibody (mAb) solutions leads to regions of different ratios of low molecular weight excipients, like buffer species or sugars, to protein. This study focused on the impact of the buffer species to mAb ratio on aggregate formation after frozen storage at −80 °C, −20 °C, and − 10 °C after 6 weeks, 6 months, and 12 months. An optimised sample preparation was established to measure Tg′ of samples with different mAb to histidine ratios via differential scanning calorimetry (DSC). After storage higher molecular weight species (HMWS) and subvisible particles (SVPs) were detected using size-exclusion chromatography (SEC) and FlowCam, respectively. For all samples, sigmoidal curves in DSC thermograms allowed to precisely determine Tg′ in formulations without glass forming sugars. Storage below Tg′ did not lead to mAb aggregation. Above Tg′, at −20 °C and − 10 °C, small changes in mAb and buffer concentration markedly impacted stability. Samples with lower mAb concentration showed increased formation of HMWS. In contrast, higher concentrated samples led to more SVPs. A shift in the mAb to histidine ratio towards mAb significantly increased overall stability. Cryoconcentration upon large-scale freezing affects mAb stability, although relative changes compared to the initial concentration are small. Storage below Tg′ completely prevents mAb aggregation and particle formation.

Potential application of low molecular weight excipients for amorphization and dissolution enhancement of carvedilol

In this study, four low molecular weight (LMW) excipients, tryptophan (TRY), phenylalanine (PHE), lysine (LYS) and saccharin (SAC) were evaluated as co-formers to generate co-amorphous systems (CAMS) by ball milling with carvedilol (CRV). Mixtures of CRV and LMW excipient in 1:0.5, 1:1 and 1:2 drug:excipient molar ratios were ball milled and analysed by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), Fourier transform (FT-IR) infrared spectroscopy and dissolution testing. CAMS were formed by milling of a mixture of CRV with TRY in 1:2 M ratio and SAC in 1:1 M ratio, while amorphization of only CRV was achieved in other mixtures with SAC. In other samples containing TRY and PHE, milling resulted in partial amorphization, while LYS was the least suitable excipient for the amorphization of CRV. Unexpectedly, the highest supersaturation of CRV was achieved from samples containing CRV and LYS in 1:1 and 1:2 M ratios, despite the absence of a significant reduction in CRV crystallinity upon milling of these samples. Increase of hydrophobic surface area caused by milling of samples with TRY and PHE and agglomeration during dissolution testing of samples containing SAC are likely causes of poor dissolution performance of mixtures containing fully or partially amorphous CRV.

Combining crystalline and polymeric excipients in API solid dispersions – Opportunity or risk?

Amorphous solid dispersions (ASDs) are often metastable against crystallization of the active pharmaceutical ingredient (API) and thus might undergo unwanted changes during storage. The crystallization tendency of ASDs is influenced by the API crystallization driving force (CDF) and the mobility of the molecules in the ASD. Low molecular weight-excipients are known to stabilize amorphous APIs in so-called co-amorphous formulations. Due to their success in stabilizing co-amorphous APIs, low-molecular weight excipients might also enhance the stability of polymeric ASDs. In this work, we investigated the potential of combined low-molecular weight excipient/polymer formulations with in-silico tools and validated the predictions with long-term stability tests of the most promising excipient/polymer combinations. The considered critical quality attributes for the ASDs were the occurrence of amorphous phase separation, API CDF, and molecular mobility in the ASD.As an example, carbamazepine/polyvinylpyrrolidone ASDs were investigated combined with the excipients fructose, lactose, sucrose, trehalose, saccharin, tryptophan, and urea. Although all excipients had a negative impact on the ASD stability, saccharin still turned out to be the most promising one. Long-term stability studies with ASDs containing either saccharin or tryptophan verified -in agreement to the predictions- that API crystallization occurred faster than in the reference ASDs without additional excipient.This work showed that the addition of crystalline excipients to polymeric ASDs might not only offer opportunities but might also bear risks for the long-term stability of the ASD, even though the crystalline excipient stabilizes the polymer-free API. Consequently, excipients should be evaluated based on the thermodynamic phase behavior of the individual mixture of API/polymer/excipient, rather than based on pure-component properties of the excipient only. In-silico predictions proposed in this work remarkably decrease the number of screening tests for identifying suitable formulation excipients.

Formulating monoclonal antibodies as powders for reconstitution at high concentration using spray-drying: Trehalose/amino acid combinations as reconstitution time reducing and stability improving formulations

To increase their stability, therapeutic (or monoclonal) antibodies (mAbs) are often formulated as solids by using a variety of drying techniques, e.g. freeze-drying, spray-drying, or spray freeze-drying. The addition of excipients is required to preserve stability of the protein during the drying process and subsequent storage of the resulting solid form. The addition of low molecular weight excipients, such as amino acids, to sugar based spray- and freeze-dried formulations has been suggested to improve the storage stability of proteins in the dried state. In this study sugars (sucrose, trehalose), amino acids (Gly, Ala, Pro, Ser, Val, Leu, Ile, Gln, His, Lys, Arg, Phe, Trp) and combinations thereof were investigated for their stabilizing effect during spray-drying and subsequent storage and for their reconstitution time reducing effect. Two IgG4 mAbs were used as model antibodies.From an initial screening study, basic and small neutral amino acids, in combination with a sugar, such as sucrose or trehalose, showed reconstitution time reducing and stabilizing properties. Arg in particular displayed excellent reconstitution and stability enhancing properties. Moreover, Arg was the only amino acid providing stabilizing properties comparable to sucrose or trehalose. Previous work by the authors described a statistically substantiated comparison between the three basic amino acids in a sugar containing formulation, albeit limited to a single concentration level [5]. Therefore, a follow-up design of experiments (DoE) study was performed to determine the optimum trehalose/amino acid content required for an optimal protein stability and reconstitution time and to compare the effects of two basic amino acids, Lys and Arg, to those of two neutral amino acids, Gly and Pro. The conducted DoE covered a wide range of trehalose (30–120 mM) and amino acid (50–150 mM) concentrations. The concentration of trehalose was found to be the main contributor to a reduction in reconstitution time and an increase in stability. Here we show that the addition of amino acids such as Gly, Pro, and Lys does not improve stability nor does it reduce the reconstitution time. Of the tested amino acids, only Arg showed a marked reduction in reconstitution time and improvement in stability compared to a trehalose. Moreover, the properties displayed by Arg could justify its application as the main stabilizer in spray-dried mAb formulations, eliminating the need for a sugar matrix altogether. But the weight ratio of stabilizer to protein was found the factor exerting the strongest overall influence on the formulation’s reconstitution time and stability. More specifically, sufficient physical stability and an acceptable reconstitution time could be obtained with a protein to stabilizer weight ratio of at least 1:1.

Aspartame as a co-former in co-amorphous systems

Co-amorphous drug delivery systems are a promising approach to improve the dissolution rate and therefore potentially the oral bioavailability of poorly-water soluble drugs. Several low molecular weight excipients, for instance amino acids, have previously been shown to stabilize the amorphous form and increase the dissolution rate of drugs. In this study, the feasibility of aspartame, a methyl ester of the aspartic acid-phenylalanine dipeptide, as a co-former was investigated and compared with the respective single amino acids, both alone and in combination. The poorly water-soluble compounds mebendazole, tadalafil and piroxicam were chosen as model drugs. In contrast to the single amino acids or the physical mixture of both, all drug-aspartame mixtures became amorphous upon 90 min of ball milling. Only a single glass transition temperature (Tg) was detected by modulated differential scanning calorimetry, which indicates that a homogeneous single-phase co-amorphous system was obtained. Powder dissolution tests showed that the dissolution rates of the drugs from drug-aspartame co-amorphous samples were increased compared to crystalline drugs. Furthermore, supersaturation was observed for the mebendazole-aspartame and tadalafil-aspartame co-amorphous systems. In conclusion, aspartame has been shown to be a promising co-former in co-amorphous systems, superior to the single amino acids or their mixtures.

Investigation of Drug-Excipient Interactions in Biclotymol Amorphous Solid Dispersions.

The effect of low molecular weight excipients on drug-excipient interactions, molecular mobility, and propensity to recrystallization of an amorphous active pharmaceutical ingredient is investigated. Two structurally related excipients (α-pentaacetylglucose and β-pentaacetylglucose), five different drug:excipient ratios (1:5, 1:2, 1:1, 2:1, and 5:1, w/w), and three different solid state characterization tools (differential scanning calorimetry, X-ray powder diffraction, and dielectric relaxation spectroscopy) were selected for the present research. Our investigation has shown that the excipient concentration and its molecular structure reveal quasi-identical molecular dynamic behavior of solid dispersions above and below the glass transition temperature. Across to complementary quantum mechanical simulations, we point out a clear indication of a strong interaction between biclotymol and the acetylated saccharides. Moreover, the thermodynamic study on these amorphous solid dispersions highlighted a stabilizing effect of α-pentaacetylglucose regardless of its quantity while an excessive concentration of β-pentaacetylglucose revealed a poor crystallization inhibition. Finally, through long-term stability studies, we also showed the limiting excipient concentration needed to stabilize our amorphous API. Herewith, the developed procedure in this paper appears to be a promising tool for solid-state characterization of complex pharmaceutical formulations.

Influence of Low-Molecular-Weight Excipients on the Phase Behavior of PVPVA64 Amorphous Solid Dispersions.

The oral bioavailability of poorly water-soluble active pharmaceutical ingredients (APIs) can be improved by the preparation of amorphous solid dispersions (ASDs) where the API is dissolved in polymeric excipients. Desired properties of such ASDs like storage stability, dissolution behavior, and processability can be optimized by additional excipients. In this work, the influence of so-called low-molecular-weight excipients (LMWEs) on the phase behavior of ASDs was investigated. Binary ASDs of an amorphous API, naproxen (NAP) or acetaminophen (APAP), embedded in poly-(vinylpyrrolidone-co-vinyl acetate) (PVPVA64) were chosen as reference systems. Polyethylene glycol 1500 (PEG1500), D-α-tocopherol polyethylene glycol 1000 succinate (TPGS1000), propylene glycol monocaprylate type II (Capryol™ 90), and propylene glycol monolaurate type I (Lauroglycol™ FCC) were used as LMWEs. The API solubility in the excipients and the glass-transition temperature of the ASDs were modeled using the Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT) and the Kwei equation, respectively, and compared to corresponding experimental data. The API solubility curves in ternary systems with 90/10wt%/wt% PVPVA64/LMWE ratios were very close to those in pure PVPVA64. However, the glass-transition temperatures of API/PVPVA64/LMWE ASDs were much lower than those of API/PVPVA64 ASDs. These effects were determined experimentally and agreed with the predictions using the PC-SAFT and Kwei models. The impact of the LMWEs on the thermodynamic stability of the ASDs is quite small while the kinetic stability is significantly decreased even by small LMWE amounts. PC-SAFT and the Kwei equation are suitable tools for predicting the influence of LMWEs on the ASD phase behavior.

Development of a screening method for co-amorphous formulations of drugs and amino acids

Using amino acids (AA) as low molecular weight excipients in the preparation of co-amorphous blends with the aim to stabilize the drug in the amorphous form have been discussed in a range of studies. However, there is currently no theoretical consensus behind which AA would be a suitable co-former for a given drug. In this work, a fast screening process to assess the co-former feasibility in co-amorphous drug-AA blends has been developed on the basis of the amorphization kinetics upon oscillatory ball milling. For this purpose, six model drugs were combined with 20 different AAs and co-milled at an equimolar ratio for different times (1, 5, 15, 30 and 60min). The degree of amorphization was then studied for the different time points by determination of the area under the curve of the diffraction peaks in X-ray powder diffraction measurements. The results of this study suggest that the choice of AA as co-formers for the formation of the co-amorphous blend could be significantly inferred after 15min of milling, since a crystallinity decrease higher than 90% after 15min resulted in successful co-amorphization in approximately 90% of the mixtures after 60min of milling. The results furthermore suggested that non-polar AAs, such as tryptophan, phenylalanine, leucine, isoleucine, methionine, valine and proline, are a good first choice in the selection of a co-former for a given drug in a co-amorphous formulation. Basic AAs appear suitable for amorphous salt formation in the case of acidic drugs. Acidic AAs however, were shown to be generally poor co-formers for co-amorphous systems.

Addition of Amino Acids to Further Stabilize Lyophilized Sucrose-Based Protein Formulations: I. Screening of 15 Amino Acids in Two Model Proteins

In small amounts, the low molecular weight excipients—sorbitol and glycerol—have been shown to stabilize lyophilized sucrose-based protein formulations. The purpose of this study was to explore the use of amino acids as low molecular weight excipients to similarly enhance stability. Model proteins, recombinant human serum albumin and α-chymotrypsin, were formulated with sucrose in combination with one of 15 amino acid additives. Each formulation was lyophilized at 1:1:0.3 (w/w) protein–sucrose–amino acid. Percent total soluble aggregate was measured by size-exclusion chromatography before and after storage at 50°C for 2 months. Classical thought might suggest that the addition of the amino acids to the sucrose-protein formulations would be destabilizing because of a decrease in the system's glass transition temperature. However, significant improvement in storage stability was observed for almost all formulations at the ratio of amino acid used. Weak correlations were found between the extent of stabilization and both amino acid molar volume and side-chain charge. The addition of amino acids at a modest level generally improves storage stability, often by more than a 50% increase, for lyophilized sucrose-based protein formulations.

Solubility of crystalline organic compounds in high and low molecular weight amorphous matrices above and below the glass transition by zero enthalpy extrapolation

Pharmaceutical applications which require knowledge of the solubility of a crystalline compound in an amorphous matrix are abundant in the literature. Several methods that allow the determination of such data have been reported, but so far have only been applicable to amorphous polymers above the glass transition of the resulting composites. The current work presents, for the first time, a reliable method for the determination of the solubility of crystalline pharmaceutical compounds in high and low molecular weight amorphous matrices at the glass transition and at room temperature (i.e. below the glass transition temperature), respectively.The solubilities of mannitol and indomethacin in polyvinyl pyrrolidone (PVP) K15 and PVP K25, respectively were measured at different temperatures. Mixtures of undissolved crystalline solute and saturated amorphous phase were obtained by annealing at a given temperature. The solubility at this temperature was then obtained by measuring the melting enthalpy of the crystalline phase, plotting it as a function of composition and extrapolating to zero enthalpy. This new method yielded results in accordance with the predictions reported in the literature.The method was also adapted for the measurement of the solubility of crystalline low molecular weight excipients in amorphous active pharmaceutical ingredients (APIs). The solubility of mannitol, glutaric acid and adipic acid in both indomethacin and sulfadimidine was experimentally determined and successfully compared with the difference between their respective calculated Hildebrand solubility parameters. As expected from the calculations, the dicarboxylic acids exhibited a high solubility in both amorphous indomethacin and sulfadimidine, whereas mannitol was almost insoluble in the same amorphous phases at room temperature.This work constitutes the first report of the methodology for determining an experimentally measured solubility for a low molecular weight crystalline solute in a low molecular weight amorphous matrix.

Polymethacrylates as crystallization inhibitors in monolayer transdermal patches containing ibuprofen

The feasibility of a monolayer patch based on polydimethylsiloxane pressure sensitive adhesive containing ibuprofen (IB) in supersaturated condition was studied. The efficacy of three low molecular weight excipients (propylene glycol, PG, Cremophor EL and Cremophor RH) and of two copolymers of methacrylic acid (Eudragit ® E, EuE, and Eudragit ® RL, EuRL) as IB crystallization inhibitors was tested. The performances of the patches were evaluated in terms of drug release and human stratum corneum and epidermis (SCE) permeation profile. The interactions between IB and the other excipients were investigated by ATR–FT-IR spectroscopy. The stability of the patches, prepared without adding crystallization inhibitors, was unsatisfactory because crystals grew in less than 1 month. Among the low molecular weight molecules, only PG inhibited the IB crystallization up to 50 days without affecting the IB skin permeation profile. The addition of EuE or EuRL in the matrices prevented drug crystallization for more than 12 months. EuE significantly reduced the IB in vitro release rate and the IB permeated amount through the SCE compared to other formulations. These phenomena are attributed to a stronger association between IB and EuE than IB and EuRL.