Development and Optimization of a New UPLC-UV/MS Method through DoE and MLR for Detecting Substandard Drug Products to Treat Tuberculosis.

Drug products used for treating tuberculosis are one of the most widely reported medicines to be classified as falsified or substandard in low- and middle-income countries, representing a major hazard to health. The aim of this study was, firstly, to develop an ultra-performance liquid chromatography (UPLC) method which is able to analyze fixed combination tablets with up to four active pharmaceutical ingredients, including isoniazid, pyrazinamide, rifampicin, and ethambutol. Secondly, we aimed to optimize it through the design of experiments and multi-linear regression based on a central composite design and to validate it according to the guidelines of the International Conference on Harmonization. The application of this tools enabled the identification of the influential factors (flow rate, formic acid, and temperature) and their effects on the studied responses (retention factor and percentage for each drug) as part of the quality by design approach. The method proved to be to be linear in the range from 5.0 to 15 µg/mL for isoniazid, pyrazinamide, and rifampicin, being precise (<1%) and accurate (97−101%). In addition, the method validated for ethambutol proved to be linear from 1.4 to 4.2 µg/mL, as well as precise (0.54%) and accurate (97.3%). The method was stability indicated for all the active pharmaceutical ingredients studied and was able to detect two substandard formulations sampled on the African market.

Design and optimization of a child-friendly dispersible tablet containing isoniazid, pyrazinamide, and rifampicin for treating tuberculosis in pediatrics.

Objective: Develop a child-friendly Fixed Dose Combination (FDC) water-dispersible tablet for Tuberculosis (TB) treatment, with 50, 150, and 75 mg of isoniazid, pyrazinamide and rifampicin respectively. This new formulation must contain the lowest number of excipients accepted for pediatrics and fulfill all the pharmacopeia requirements.Significance: At present, there is no adequate market dosage form available for children. There is, however, one in a prequalification phase by the World Health Organization but its composition contains excipients which may not be suitable for pediatrics. Therefore, this new formulation would cover this therapeutic gap.Methods: A factorial design, based on three quantitative factors (compression force and concentration of AcDiSol® and Explosol®) at three levels each, was performed to elucidate their influence over disintegration time and friability. In addition, the influence of the press speed on disintegration time, friability, tensile strength, fineness of dispersion and content uniformity over the target tablet was tested. A stability test was done following ICH guideline for accelerated conditions.Results: Tablets developed with 9% w/w of Explosol® and a compression force of 16 kN disintegrated in less than 3 min and showed a friability below 1% when 15-mm punches were used. The tableting process could be done up to 25 and 50 cycles/minute ensuring good quality attributes when 15 and 12-mm punches were used, respectively. All APIs remained inside the limit of ± 5% of drug content till 6 months of storage.Conclusion: A high-quality child-friendly FDC water-dispersible tablet was developed improving the treatment of TB in pediatric.

A High-Demanding Strategy to Ensure the Highest Quality Standards of Oral Liquid Individualized Medicines for Pediatric Use.

Individualized medicines for pediatrics are a useful alternative if there is no correct dosage marketed for this segment (easy to swallow, adequate volume and content, correct composition for pediatrics, good organoleptic properties, etc.). Its validation process must ensure quality testing: its content uniformity, physical (homogeneity after shaking), chemical, and microbiological stability. Some of these attributes are checked by the recommendations of European Pharmacopoeia (Ph. Eur.), International Conference of Harmonization (ICH), and National Formularies but others are not. The aim of this study is to develop a general high-demanding strategy to ensure the final quality of liquid dosage forms testing and developing standard operating processes (SOPs) for the elaboration of individualized oral liquid medicines for pediatric use. Furosemide was used as an example of the validation of an individualized liquid solution for pediatric use. Three SOPs were selected according to their composition and the recommendations of liquid dosage forms for pediatric use. Quality attributes according to National Formularies, Ph. Eur., and ICH were tested: pH, organoleptic properties, uniformity of mass of delivered dose from multidose containers, and chemical stability. In this study, a general high-demanding strategy was elaborated to validate oral liquid dosage forms, including validation of the analytical method used to test their quality. A second part focuses on the elaboration of liquid formulations for pediatrics with the highest standards of quality taking into account CQAs that were not contemplated by official compendial such as content uniformity and physical stability.

Development of a novel physico-chemically and microbiologically stable oral solution of flecainide for pediatrics.

There is as yet no commercialized preparation for oral administration of flecainide acetate (FA) to children. In such cases, manipulation of commercial tablets is the usual practice in pharmacy services of hospitals and compounding pharmacies, to provide a suitable dosage form for this vulnerable pediatric population group. In this study, we have formulated FA as an oral solution, as an alternative to the suspension elaborated from commercial tablets. Due to this sensitivity of young patients, we have used the pure active pharmaceutical ingredient (API) and the lowest permitted levels of pediatric excipients. Despite being a highly soluble API, only one of the formulations appears as a transparent solution due to complete FA solubilization. The proposed formulation is physico-chemically and microbiologically stable and the mass and dose uniformity is appropriate for 30 days' storage at 25 °C.

Formulation design of oral pediatric Acetazolamide suspension: dose uniformity and physico-chemical stability study.

CONTEXT: The formulation of an active pharmaceutical ingredient (API) as oral solution or suspension in pediatrics is a habitual practice, due to the non-existence of many commercialized medicines in pediatric doses. It is also the simplest way to prepare and administer them to this vulnerable population. The design of a formulation that assures the dose and the system stability depends on the physico-chemical properties of the API. OBJECTIVE: In this study, we formulate a class IV API, Acetazolamide (AZM) as suspension for oral administration to pediatric population. The suspension must comply attributes of quality, safety and efficacy for this route of administration. MATERIALS AND METHODS: We use simple compounding procedures, as well as fewer pure excipients, as recommended for children. Mass and uniformity content assays and physical and chemical stability studies were performed. To quantify the API an UPLC method was used. RESULTS AND DISCUSSION: We verified the physico-chemical stability of the suspensions and that they passed the mass test of the European Pharmacopeia (EP), but not the dose uniformity test. CONCLUSIONS: This reveals that AZM must be formulated as liquid forms with a more complex system of excipients (not usually indicated in pediatrics), or otherwise solid forms capable of assuring uniformity of mass and dose for every dosage unit.

Development of an ultra high performance liquid chromatography method for determining triamcinolone acetonide in hydrogels using the design of experiments/design space strategy in combination with process capability index.

An ultra high performance liquid chromatography method was developed and validated for the quantitation of triamcinolone acetonide in an injectable ophthalmic hydrogel to determine the contribution of analytical method error in the content uniformity measurement. During the development phase, the design of experiments/design space strategy was used. For this, the free R-program was used as a commercial software alternative, a fast efficient tool for data analysis. The process capability index was used to find the permitted level of variation for each factor and to define the design space. All these aspects were analyzed and discussed under different experimental conditions by the Monte Carlo simulation method. Second, a pre-study validation procedure was performed in accordance with the International Conference on Harmonization guidelines. The validated method was applied for the determination of uniformity of dosage units and the reasons for variability (inhomogeneity and the analytical method error) were analyzed based on the overall uncertainty.

Quality assessment of oral antimalarial and antiretroviral medicines used by public health systems in Sahel countries.

Malaria and Human Immunodeficiency Virus infections are among the top 10 causes of death in low income countries. Furthermore, many medicines used in these treatment areas are substandard, which contributes to the high death rate. Using a monitoring system to identify substandard and falsified medicines, the study aims to evaluate the quality of antimalarial and antiretroviral medicines in Sahel countries, assessing site conditions, compliance of medicines with pharmacopoeia tests, formulation equivalence with a reference medicine, and the influence of climate on quality attributes. Ultra Performance Liquid Chromatography methods for eight active pharmaceutical ingredients were validated following the International Conference for Harmonization guideline for its detection and quantification. Quality control consists of visual inspections to detect any misinformation or imperfections and pharmacopeial testing to determine the quality of pharmaceutical products. Medicines which complied with uniformity dosage units and dissolution tests were stored under accelerated conditions for 6 months. Artemether/Lumefantrine and Lopinavir/Ritonavir formulations failed uniformity dosage units and disintegration tests respectively, detecting a total of 28.6% substandard medicines. After 6 months stored under accelerated conditions (40 °C // 75% relative humidity) simulating climatic conditions in Sahel countries, some medicines failed pharmacopeia tests. It demonstrated the influence of these two factors in their quality attributes. This study emphasizes the need of certified quality control laboratories as well as the need for regulatory systems to maintain standards in pharmaceutical manufacturing and distribution in these countries, especially when medicines are transported to rural areas where these climatic conditions are harsher.

Revolutionizing Three-Dimensional Printing: Enhancing Quality Assurance and Point-of-Care Integration through Instrumentation.

Three-dimensional printing in the field of additive manufacturing shows potential for customized medicines and solving gaps in paediatric formulations. Despite successful clinical trials, 3D printing use in pharmaceutical point-of-care is limited by regulatory loopholes and a lack of Pharmacopoeia guidelines to ensure quality. Semi-solid extrusion is a 3D printing technology that stands out for its versatility, but understanding the fluid dynamics of the semi-solid mass is critical. The aim of this research is to look into the advantages of instrumenting a 3D printer with a semi-solid extrusion motor-driven printhead, which is able to record the printing pressure over time, for in situ characterization of the semi-solid mass and quality evaluation of dosage forms. Four formulations using hydrochlorothiazide as the active pharmaceutical ingredient and several excipients were used. Their flow properties were studied at different printing speeds and temperatures using traditional techniques (rheometer and Texture Analyzer) and the proposed semi-solid extrusion motor-driven printhead incorporated into a printing platform. In addition, the influence of printing speed in the printing process was also evaluated by the study of printing pressure and printlet quality. The results demonstrated the similarities between the use of a Texture Analyzer and the semi-solid extrusion motor-driven. However, the latter enables temperature selection and printing speed in accordance with the printing process which are critical printing parameters. In addition, due to the incorporation of a sensor, it was possible to conclude, for the first time, that there is a link between changes in essential printing parameters like printing speed or formulations and variations in printing pressure and printlet quality attributes such as the energy require to obtain a single dosage unit, weight or diameter. This breakthrough holds a lot of potential for assuring the quality of 3D printing dosage forms and paving the way for their future incorporation into point-of-care settings.

Characterization and Validation of a New 3D Printing Ink for Reducing Therapeutic Gap in Pediatrics through Individualized Medicines.

3D printing technology can be used to develop individualized medicines in hospitals and pharmacies, allowing a high degree of personalization and the possibility to adjust the dose of the API based on the quantity of material extruded. The main goal of incorporating this technology is to have a stock of API-load print cartridges that could be used at different storage times and for different patients. However, it is necessary to study the extrudability, stability, and buildability of these print cartridges during storage time. A paste-like formulation containing hydrochlorothiazide as a model drug was prepared and distributed in five print cartridges, each of which was studied for different storage times (0 h-72 h) and conditions, for repeated use on different days. For each print cartridge, an extrudability analysis was performed, and subsequently, 100 unit forms of 10 mg hydrochlorothiazide were printed. Finally, various dosage units containing different doses were printed, taking into account the optimized printing parameters based on the results of the extrudability analysis carried out previously. An appropriate methodology for the rapid development of appropriate SSE 3DP inks for pediatrics was established and evaluated. The extrudability analysis and several parameters allowed the detection of changes in the mechanical behavior of the printing inks, the pressure interval of the steady flow, and the selection of the volume of ink to be extruded to obtain each of the required doses. The print cartridges were stable for up to 72 h after processing, and orodispersible printlets containing 6 mg to 24 mg of hydrochlorothiazide can be produced using the same print cartridge and during the same printing process with guaranteed content and chemical stability. The proposed workflow for the development of new printing inks containing APIs will allow the optimization of feedstock material and human resources in pharmacy or hospital pharmacy services, thus speeding up their development and reducing costs.

A pharmaceutical monitoring system to assess the quality of antituberculosis drug products used in Mauritania.

The quality of drug products may be affected from manufacture to dispensing, particularly at high temperature and humidity as in Mauritania. This country is not included in the World Health Organization reports on poor quality products due to the lack of a qualified laboratory and monitoring system. Ensuring the quality of medicine is even more relevant in the case of diseases such as Tuberculosis, due to its high prevalence, complex treatment and continuous bacterial resistance. The aim was to develop a monitoring system to assess the quality of antituberculosis drugs products, by the substandard detection based on European and United States Pharmacopeial recommendations regarding quality control. In addition to studying the influence of accelerated storage conditions (40 ± 2°C/75 ± 5% relative humidity) on their qualities and comparing the dissolution profiles to contrast the quality. 18 antituberculosis drug products were taken from Europe and Mauritania, and quality was studied through visual inspection and according to the compliance of the mass uniformity, uniformity of dosage units, dissolution, disintegration and friability pharmacopeial tests. Furthermore, a dissolution profile comparison was carried out to examine quality. A stability study was conducted to assess the influence of climatic conditions on the content and the dissolved amount of the active pharmaceutical ingredients, which were determined by an ultra-performance liquid chromatography system. As result, 69.3% of 13 Mauritanian formulations had a substandard quality mainly due to non-compliance with the test for friability or content uniformity of these medicines. All European drug products complied with pharmacopeia specifications. In addition, storage conditions affected the dissolution rate of ethambutol and the uniformity of the 4 antituberculosis combination drug products.

Integrating pressure sensor control into semi-solid extrusion 3D printing to optimize medicine manufacturing.

Semi-solid extrusion (SSE) is a three-dimensional printing (3DP) process that involves the extrusion of a gel or paste-like material via a syringe-based printhead to create the desired object. In pharmaceuticals, SSE 3DP has already been used to manufacture formulations for human clinical studies. To further support its clinical adoption, the use of a pressure sensor may provide information on the printability of the feedstock material in situ and under the exact printing conditions for quality control purposes. This study aimed to integrate a pressure sensor in an SSE pharmaceutical 3D printer for both material characterization and as a process analytical technology (PAT) to monitor the printing process. In this study, three materials of different consistency were tested (soft vaseline, gel-like mass and paste-like mass) under 12 different conditions, by changing flow rate, temperature, or nozzle diameter. The use of a pressure sensor allowed, for the first time, the characterization of rheological properties of the inks, which exhibited temperature-dependent, plastic and viscoelastic behaviours. Controlling critical material attributes and 3D printing process parameters may allow a quality by design (QbD) approach to facilitate a high-fidelity 3D printing process critical for the future of personalized medicine.

Supercritical CO2 technology for one-pot foaming and sterilization of polymeric scaffolds for bone regeneration.

Sterilization is a quite challenging step in the development of novel polymeric scaffolds for regenerative medicine since conventional sterilization techniques may significantly alter their morphological and physicochemical properties. Supercritical (sc) sterilization, i.e. the use of scCO2 as a sterilizing agent, emerges as a promising sterilization method due to the mild operational conditions and excellent penetration capability. In this work, a scCO2 protocol was implemented for the one-pot preparation and sterilization of poly(ε-caprolactone) (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds. The sterilization conditions were established after screening against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) vegetative bacteria and spores of Bacillus stearothermophilus, Bacillus pumilus and Bacillus atrophaeus. The transition from the sterilization conditions (140 bar, 39 °C) to the compressed foaming (60 bar, 26 °C) was performed through controlled depressurization (3.2 bar/min) and CO2 liquid flow. Controlled depressurization/pressurization cycles were subsequently applied. Using this scCO2 technology toolbox, sterile scaffolds of well-controlled pore architecture were obtained. This sterilization procedure successfully achieved not only SAL-6 against well-known resistant bacteria endospores but also improved the scaffold morphologies compared to standard gamma radiation sterilization procedures.

Stability Study of Isoniazid and Rifampicin Oral Solutions Using Hydroxypropyl-Β-Cyclodextrin to Treat Tuberculosis in Paediatrics.

(1) Background: First-line antituberculosis treatment in paediatrics entails the administration of Isoniazid, Pyrazinamide, and Rifampicin. This study examines the possibility of developing a combined dose liquid formulation for oral use that would facilitate dose adjustment and adherence to treatment for younger children. (2) Methods: The active pharmaceutical ingredients stability under in vitro paediatric digestive pH conditions have been checked. The samples were studied as individual or fixed combined paediatric dosages to determine the pH of maximum stability. The use of hydroxypropyl-ß-cyclodextrin to improve Rifampicin solubility and the use of ascorbic acid to increase the stability of the formulation have been studied. (3) Results: Maximum stability of combined doses was determined at pH 7.4, and maximum complexation at pH 8.0. Taking this into account, formulations presented the minimum dose of two active pharmaceutical ingredients dissolved. The addition of ascorbic acid at 0.1% w/v enables the detection of a higher remaining quantity of both drugs after three days of storage at 5 °C. (4) Conclusions: a formulation which combines the minimum paediatric dosages dissolved recommended by WHO for Isoniazid and Rifampicin has been developed. Future assays are needed to prolong the stability of the formulation with the aim of incorporating Pyrazinamide to the solution.

Effectiveness of Antimicrobial Preservation of Extemporaneous Diluted Simple Syrup Vehicles for Pediatrics.

OBJECTIVES: Extemporaneous or magistral formulation of active pharmaceutical ingredients using traditional compounding techniques is a common practice when no commercial form is available for pediatrics. For this vulnerable group of patients, the formulation must be prepared with the minimum quantity and lowest proportion of excipients approved for pediatrics, avoiding the use of preservatives. Often the vehicles used for these preparations are dilutions of simple syrup with water. The objective of this study is to assess the effectiveness of antimicrobial preservation in simple syrup diluted with aqua conservans (conserved water), without propylene glycol or with a reduced proportion of parabens. METHODS: The European Pharmacopoeia test of efficacy of antimicrobial preservation was applied to 5 trial vehicles prepared with simple syrup diluted with water. RESULTS: Simple syrup is stable during 14 days. Vehicles prepared with simple syrup diluted with purified water did not meet the microbiological quality criteria, but when they are diluted with water that incorporates propylene glycol and parabens (aqua conservans), then they meet the criteria. In addition, if the water is prepared with parabens and without propylene glycol, the criteria for the dilution are met. Nevertheless, if the dilution is done with water prepared with an insufficient proportion of parabens to act as preservatives, the dilution does not meet the pharmacopoeia microbiological criteria. CONCLUSIONS: Dilution of simple syrup (50:50 v/v) to prepare a vehicle for extemporaneous or magistral preparation is microbiologically safe when water with methylparaben and propylparaben is used in a proportion of 0.08% and 0.02% (w/w), respectively, avoiding the use of propylene glycol as a solvent and thus its toxic effects in pediatrics.

Structure-Performance Relationships of Temperature-Responsive PLGA-PEG-PLGA Gels for Sustained Release of Bone Morphogenetic Protein-2.

PLGA (poly(lactic-co-glycolic) acid)-PEG (polyethylene glycol)-PLGA synthesis conditions have an impact on the physicochemical features of the copolymer and its usefulness as biomaterial. This study reports on an analysis of the composition and structural properties of PLGA-PEG-PLGA copolymers applying a variety of analytical techniques. Viscoelastic properties and particularly the temperature-responsive behavior of PLGA-PEG-PLGA showed a marked dependence on copolymer structural features. Physicochemical and biological properties, such as bioadhesion, biocompatibility and cell viability, of the raw copolymers and their gels were also evaluated. The most promising copolymer was chosen to formulate the osteoinductive protein bone morphogenetic protein-2 (125I-BMP-2), and the ability of its gels to sustain the release both in vitro and in vivo was monitored in situ using a gamma counter. In vitro diffusion studies were carried out using a bioinspired set-up that included a biorelevant receptor medium. In vivo release tests after implantation in a critical-size calvarial defect model showed an important burst, but then the release fitted well to the square-root kinetics. Importantly, the release rate constants recorded in vitro and in vivo matched each other suggesting close in vitro-in vivo correlation. Overall, the information gathered opens new perspectives in the biomedical application of these temperature-sensitive materials.

Pre-study and in-study validation of a size-exclusion chromatography method with different detection modes for the analysis of monoclonal antibody aggregates.

Size exclusion chromatography (SEC) with different detection modes was assessed as a means to characterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics. The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and ß-expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggregation rate for 5°C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life.

Fitting bevacizumab aggregation kinetic data with the Finke-Watzky two-step model: Effect of thermal and mechanical stress.

Size exclusion chromatography with light scattering detection (SEC-MALLS) was assessed as a means to characterize the type of bevacizumab aggregates that form under mechanical and thermal stress, quantitatively monitoring the aggregation kinetics. The analytical method was monitored and verified during routine use at two levels: (1) the "pre-study" validation shows that the method is specific, linear, accurate, precise, robust and stability indicating; (2) the "in-study" validation was verified by inserting quality control samples and the use of control charts, indicating that the analytical method is in statistical control and stable. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model, but the quality of the fit can be considered poor (R(2)>0.96), especially at higher temperatures. This indicates that the order of the reaction could not be reliably determined, suggesting a different degradation mechanism. The kinetic data set also fit the minimalistic Finke-Watzky (F-W) 2-step model, with an excellent quality of fit (R(2)>0.99), yielding the first quantitative rate constant for the steps of nucleation and growth in bevacizumab aggregation. The bevacizumab pharmaceutical preparation contains (initially) dimers, approximately 1.6% of bevacizumab total concentration, and the effect on aggregation kinetics of seeding was analyzed using the F-W 2-step model assuming [B]0≠0 (for the seeded case). The results suggested that the seeding had no impact on aggregation kinetics. Furthermore, the Arrhenius equation cannot be used to extrapolate the shelf-life since no linear temperature dependence of the rate constant was found within the temperature range. Although the real-time stability data provides the basis for determining the product shelf-life, predictive methodologies such as Vogel-Tammann-Fulcher (VFT) or the Arrhenius approach can be misleading and result in overestimates of the product shelf-life. However, they can be successfully applied to fixing the lower and upper limits of the aggregation rate, i.e. the best and worst-case scenarios regarding the aggregation potential of the product. In conclusion, the present study evaluates the first application of the F-W 2-step model to fitting and interpretation of experimental aggregation data for bevacizumab pharmaceutical preparations, using SEC-MALLS in this context.

Biodegradable laminar implants for sustained release of recombinant human growth hormone.

Due to its short half-life, renal toxicity and necessity for daily subcutaneous injections, recombinant human growth hormone (rhGH) would best be administered in a controlled release formulation. One approach to this is the use of biodegradable laminar implants based on poly(lactic/glycolic) acid. Two PLGA laminar implant formulations (10% w/w), F(1) and F(2), were designed and statistically compared with a conventional commercial injectable (Norditropin). The following variables were chosen as responses: body weight, organ weights (heart, spleen and thymus), and tibia weights and dimensions. After statistical analysis, the most sensitive responses for detecting differences between formulations were body weight, tibia length and organ weights. Initial in vitro studies of these formulations showed an incomplete gradual release (after 15 days 40% rhGH released from F(1) and 60% from F(2)) and in vivo the best results were obtained with F(1). This formulation showed the best growth curve and the highest values of all the above responses. In conclusion, this type of formulation provides a sustained release of rhGH for at least 15 days and a greater efficacy than the frequent administration of the conventional injectable.

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine.

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was perforrmed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600-18,000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.

Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins.

The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and/or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates. Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution. The purpose of the current review is to describe and discuss the capability of the SEC/LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor/ligands. For this, the "two or three detector" methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes. Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.