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    Early Assessment of Pharmaceutical Formulations using OptiPharma Kit: A case study on Proprietary Pharmaceutical Protein

    Early Assessment of Pharmaceutical Formulations using OptiPharma Kit: A case study on Proprietary Pharmaceutical Protein

    Keywords: Protein Solubilization, Pharmaceutical Formulation, Formulation Development, Protein Aggregation, Protein Physical Stability

    Our previous blog describes the use of OptiPharma kit on α-chymotrypsin, commercially available protein. Here we are presenting a formulation study on a proprietary pharmaceutical protein (protein X, molecular weight of 70 kDa) using the OptiPharma Kit III. Figure 1 shows an overview of experimental procedure using an OptiPharma kit. In this experiment, a protein solution was mixed with each OptiPharma formulation and incubated for 4 hours at room temperature. The mixtures were then transferred to 96-well format filter plate and centrifuged. The amount of protein in the filtrates was detected at the wavelength of 280 nm using a UV/Vis plate reader. Detailed method can be found in OptiPharma Quickstart Guide and OptiPharma Manual.

    OptiPharma kit

    Figure 1: General experimental steps to formulate and solubilize protein using OptiPharma kit.

    Figure 2 shows the result from OptiPharma Kit III for protein X. The plots were generated using OptiPharma Dashboard. This Dashboard is available with the purchased kit. Data in Figure 2 suggests that buffers at pH 6.0 to 7.5 (except Histidine and Tris buffers) may be good buffers for the protein. This result confirms an independent study that Tris buffer is detrimental to protein X. Comparing the performance of additives in various buffers, poloxamer 188 and sodium bisulfate may hinder protein solubility in certain buffers. Amino acids and sugars seem to support protein X solubility in various buffers. Figure 3 shows the ‘positive’ effect of Arg-Glu 50/50 and ‘negative’ effect of poloxamer 188 in buffers of different pH. There are several additive-buffer combinations that would be good candidates for solubilizing protein X. Arg-Glu 50/50 in buffers pH 6.0-6.5 (preferably citrate buffer) would be good starting formulation for protein X.

    Table 1 lists nine best additives out of 40 candidate additives for formulating Protein X in solutions using HSCTM Technology. HSCTM Technology, SolubleBioScience’s core formulation technology, is a high-throughput protein formulation method based on self-interaction chromatography. Self-interaction chromatography allows the measurement of protein-protein interactions in the form of second virial coefficient, B22. High B22 values indicate enhanced protein solubility, while low B22 values indicate propensity to protein aggregation in a solution. The data obtained from OptiPharma kit are in agreement with data obtained using HSC method.  From HSC method, the B22 values for the two poor performing additives, i.e. poloxamer 188 and sodium bisulfate, are 1.0 and 0.3 B units, respectively, compared to a range of 1.6 to 2.6 for the best performing additives.

    solublebioscience-blog-Figure-1

    solublebioscience-blog-Figure-2

    Figure 2: Percent elution of Protein X in various buffers in the presence of pharmaceutical excipients. Percent elution is a ratio of protein amount in filtrate and original amount of protein before filtration. Note the value less than 0% or more than 100% is experimental errors.

    Arg-Glu 50/50

    Figure 3: Percent elution of Protein X in buffers of different pH in the presence of Arg-Glu 50/50 (left) and Poloxamer 188 (right).

    Table 1: Best additives for formulating protein X as suggested from HSCTM Method

    Additive Measured B22 Value (mol * mL / g2 * 10-4)
    1. Citric acid 2.6
    2. Arg/Glu 50/50 2.5
    3. NaCl 2.1
    4. Dextrose 2.0
    5. Sucrose 2.0
    6. EDTA 1.8
    7. Arginine-HCl 1.7
    8. Glycine 1.6
    9. Succinic acid 1.6

    Note: B22 values calculated for solutions containing individual 40 additives ranged from 0.1 to 2.6.

    In conclusion, OptiPharma Kit provides a rough estimate of best additive and/or best pH/buffer for first order of formulation development. Using an OptiPharma kit, a formulation scientist can reduce several weeks of experimental work into a few hours of work combined with stress period. The OptiPharma kit is designed as an initial formulation screen. The experimenter is expected to follow up this initial screen with a characterization of biophysical properties in the solutions to confirm the findings.

    Identifying Pharmaceutical Excipients and Solution Conditions for α-Chymotrypsin using OptiPharma Kit

    Identifying Pharmaceutical Excipients and Solution Conditions for α-Chymotrypsin using OptiPharma Kit

    Formulation development is one of the critical steps in developing a protein as a therapeutic product. Maintaining the integrity of purified protein during pharmaceutical processing, storage, handling, and delivery to patient is a major challenge. There are many solution conditions (e.g. buffer pH, ionic charge, concentration, etc.) and pharmaceutical excipients which one can choose and combine to achieve the best condition for a solubilized protein. This process is often times very time consuming and costly.

    SolubleBioScience’s OptiPharma kit provides high throughput screening method to identify key pharmaceutical solution conditions and ingredients to solubilize and stabilize a protein of interest. OptiPharma Protein Solubility Screening Kit contains a combination of 10 types of buffers (pH varies from 3 to 8.5) and 13 pharmaceutical excipients (salts, amino acids, sugars, surfactants, preservatives). A total of 93 different formulations in a 96-well format can be tested in single, label-free experimental setup. The remaining 3 wells are for experiment controls. All ingredients contained in the OptiPharma kit are proven pharmaceutical excipients and buffers.

    OptiPharma applies a filtration method to separate soluble protein molecules from aggregated protein molecules. Protein is detected in formulations that promote solubility after passing through a specific molecular weight cut-off filter. While aggregated protein, due to sub-optimal formulations, is retained on the filter. Figure 1 provides an overview of experimental steps. Detailed method can be found in OptiPharma Quickstart Guide and OptiPharma Manual.

    OptiPharma kit

    Figure 1: General experimental steps to formulate and solubilize protein using OptiPharma kit.

    There are four types of OptiPharma kit. Each type is specific to a range of molecular weight cut-off filter. Here, we used OptiPharma II for solubilization study of α-chymotrypsin (from bovine pancreas, Mw. ca. 25kDa). Protein was mixed with each formulation and incubated for 4 hours at room temperature. The mixtures were then transferred to 96-well format filter plate and centrifuged. Protein was detected at the wavelength of 280 nm using a plate reader.

    Percent elution of α-chymotrypsin

    Percent elution of α-chymotrypsin

    Figure 2: Percent elution of α-chymotrypsin in various buffers in the presence of pharmaceutical excipients. Percent elution is a ratio of protein amount in filtrate and original amount of protein before filtration. Note the values larger than 100% are due to experimental errors.

    Data in Figure 2 provide several insights into solution properties that are amenable to solubilizing α-chymotrypsin. Sodium citrate buffer pH 6.5, potassium phosphate buffer pH 7.0 and Na/K buffer pH 7.5 are likely good buffers for stabilizing α-chymotrypsin in a solution. On the other hand, one may want to stay away from buffers with pH less than 6.5. The result suggests that Arg-Glu 50/50 could be key excipient in protein solubilization. In buffers pH 6.5 to 7.5, most excipients are not hindering protein solubility, except poloxamer 188 and sodium bisulfate.

    In conclusion, OptiPharma kit provides high-throughput assessment of protein behavior in multiple solution conditions in a short period of time. Using an OptiPharma kit, a formulation scientist can reduce several weeks of trial-and-error experimental work into a few hours of work combined with stress period. The OptiPharma kit is designed as an initial formulation screen. The experimenter is expected to follow up this initial screen with a characterization of biophysical properties in the solutions to confirm the findings.

    High-Throughput Method to Formulate and Solubilize Proteins using SolubleBioScience Optisol Kit

    High-Throughput Method to Formulate and Solubilize Proteins using SolubleBioScience Optisol Kit

    A major hurdle for biologics development is the determination of a formulation that keeps a protein soluble. SolubleBioScience OptiSol Protein Solubilization Screening Kit (96-well format) contains a systematically varied array of buffers (from pH 3 to pH 10) and a series of solubility enhancers (salts, amino acids, sugars, polyols, reducing agents). Parallel filtration of the protein through multimer-excluding MWCO filters enables rapid identification of solubilizing formulations by a simple comparison of elution concentrations. A total of 90 different formulations with solubility enhancers are tested in a single, label-free experiment within 4 hours or less.

    OptiSol kit for formulating

    Figure 1: General experimental steps to formulate and solubilize protein using Optisol kit1.

    Here we explore the utility of OptiSol kit for formulating L-glutamic dehydrogenase (LGDH) in solution. Experimental steps were performed as described in Figure 1. Depending on study goals, one can choose to stress the sample using various methods (i.e. temperature, shear force, chemical exposure, etc.). In this case, we incubated the protein-reagent mixtures at room temperature for several hours. Four types of Optisol Kits are available for four different ranges of protein molecular weights. Solubilized protein molecules will be found in the filtrates. Formulations that are detrimental to protein solubilization will result in aggregated protein being retained on the filter membrane. Various detection assays can be employed to qualitatively or quantitatively analyze the amount of protein in the filtrates. In this case study, UV280 absorbance using a plate reader was employed.

    LGDH

    Figure 2: Percent elution of LGDH with respect to reagents in Optisol IV kit. Percent elution is a ratio of protein amount in filtrate and original amount of protein before filtration. Data were from triplicate experiments.

    Optisol IV kit was utilized to screen LGDH (MW: ~310 kDa). Figure 2 shows percent elutions of LGDH in Optisol formulations ranked from maximum elution to minimum elution. The top best formulations are tabulated in Table 1, in comparison to storage formulation (SB). The result suggests that polysorbate 20, glycerol, and TMAO may be suitable additives that promote LGDH solubility in solution. Data also show that various buffers and pHs can be used to solubilize LGDH.

    formulations for LGDH

    Table 1: Top formulations for LGDH and Dynamic Light Scattering (DLS) data of LGDH in respective formulations

    This study shows that Optisol kit can provide high-throughput analysis of multiple formulations in a short period of time. This is very advantageous at various steps of biopharmaceutical developments in identifying key pharmaceutical excipients to go forward with or to avoid.

    We further analyzed the properties of LGDH in top formulations using Dynamic Light Scattering (DLS) and self-interaction chromatography (SIC) methods. DLS provides insights into molecular properties of protein in solutions, i.e. hydrodynamic radius (Rh), aggregation behavior, etc. Table 1 shows that some positive formulations resulted in protein instability after a period of 1 week. We applied SIC2 method to obtain B22 (second virial coefficient) values of LGDH in some top formulations. Positive values indicate better solubility, while negative values indicate poor solubility. Data in Table 2 shows that the selected formulations performed better than storage buffer in solubilizing LGDH in solution. The addition of 1 %w/v of polysorbate into storage buffer formulation resulted in improved formulation for LGDH. Our results show that OptiSol kit provides a reliable high-throughput method for identifying solubilizing protein formulations.

    Table 2: B22 values of LGDH in selected formulations

    References

    1. OptiSolTM Protein Solubility Screening Kit Application Manual. SolubleBioScience Inc.
    2. Johnson, D. H, et al. High-Throughput Self-Interaction Chromatography: Applications in Protein Formulation Prediction. Pharmaceutical Research 26 (2): 296-301 (2009).
    Formulation Of Biosimilars

    Formulation Of Biosimilars

    Biosimilars are the buzz word du jour in the biopharma arena; however, the development of biosimilars that will truly provide cost savings that the end consumer typically expects of a “generic” drug product is the real challenge due in part to the following reasons:

    • The lack of a centralized listing of biological patents (like the Orange Book) and company analysis is kept confidential
    • Platform patents protect broad groups of products, e.g. humanized antibodies, which are not product specific
    • And formulation patents
    8 Top Tips for Improving In Vitro Translation

    8 Top Tips for Improving In Vitro Translation

    If you need a eukaryotic protein for functional studies, it makes sense to overexpress it in a cell-free system, which allows conditions closer to native including post-translational modifications. However, expressing protein in vitro does not exclude two major problems – low yield and sometimes even protein aggregation. Here are 8 tips to help you overcoming these problems.
    2013-06-15-improving_protein_yield_in_vitro_expression_lg
    Figure 1. Flowchart for improving protein expression yield in vitro


    1. Codon Usage. It may seem obvious, but do check your template design: make sure you have minimum of   codons that require rare cognate tRNAs, failing to do this may significantly reduce protein production (1).  Gene synthesis is relatively inexpensive now.

    2. Related Expression System. In general, it makes sense to express your protein in the system close to the organism it’s been cloned from, i.e. wheat germ (WG) if you are working with a plant protein, rabbit reticulocyte lysate (RRL) for animal proteins. However, you may discover that your mammalian protein is expressed better in WG: investment in small aliquots of different types of extracts can save you a lot of time and money.

    3. Pure Template. Make sure that your template, whether it’s DNA or RNA, is clean. Even small quantities of chaotropic salts, phenol, ethanol and other impurities can significantly reduce yield.

    4. Optimize Salts.  Try different potassium and magnesium concentrations in the reaction, just as with PCR different templates have different optimal ions concentration, in vitro transcription and translation have different optimums.

    5. Optimize Temperature. Try lowering the incubation temperature by 5 C. This slows down the transcription from sometimes too strong phage promoters, improving the coupling between transcription and translation. This prevents mRNA degradation and allows more time for the nascent protein folding, reducing chances of incorrect folding and subsequent aggregation. Lowering the temperature has been shown to improve yields of active protein (2).

    “Add DIY mRNA” system

    6. Functional Message. Make sure that you mRNA is functional. Checking the spectrum and concentration on a spectrometer is not enough; you have to run your RNA on a PAGE to ensure its integrity.

    7. Template Concentration. Sometimes RNA concentration is a limiting factor – try increasing the amount of RNA in the translation reaction (3).

    Coupled transcription-translation system

    8. Folding Assistance. In some cases aggregated protein is a mix of intermediates in folding pathway. A number of proteins require chaperones’ assistance for folding. Adding chaperones to the cell-free extract   will prevent the aggregates accumulation (3).

    Literature:

    1. Hatfield GW, & Roth DA (2007). Optimizing scaleup yield for protein production: Computationally Optimized DNA Assembly (CODA) and Translation Engineering. Biotechnology annual review, 13, 27-42 PMID: 17875472
    2. Iskakova MB, Szaflarski W, Dreyfus M, Remme J, & Nierhaus KH (2006). Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins. Nucleic acids research, 34 (19) PMID: 17038334
    3. Jun SY, Kang SH, & Lee KH (2008). Continuous-exchange cell-free protein synthesis using PCR-generated DNA and an RNase E-deficient extract. BioTechniques, 44 (3), 387-91 PMID: 18361792
    What Insects Did For Glycoprotein Purification

    What Insects Did For Glycoprotein Purification

    A large number of biotechnologically relevant proteins, including enzymes, hormones, cytokines, clotting factors, monoclonal antibodies are biologically active only in the glycoprotein form (1).

    Glycoproteins – proteins with covalently attached oligosaccharide chains – are not the easiest type of proteins to work with. The oligosaccharide (glycan) part, important for the correct folding and/or biological activity of the purified protein, is attached to the synthesized polypeptide posttranslationally. This means that the conventional purification method such as production and purification in E.coli does not work, because bacteria lack posttranslational mechanisms necessary for the sugars attachment to the polypeptide chain. The expression of glycoproteins without their oligosaccharide counterpart, however, often leads to the protein aggregation and their abnormal targeting if eukaryotic cells were used for the production (2).

    2013-06-04-fall_armyworm_protein_production_2

    Figure1 Fall armyworm – what can it be good for? (Picture from Wikimedia Commons )

    Glycoprotein production in human cell culture, while overcoming the problem of glycoprotein aggregation and incorrect targeting, creates a new challenge – human-derived products are subject to very strict quality control on all production stages, including the serum used for human cell culture. After all, they need to pass the European Medicines Agency and U.S. Food and Drug Administration requirements for the recombinant products.
    Figure 2. Fall armyworm cell line Sf21, one of the most popular insect cell lines for recombinant protein production. (Picture from Wikimedia Commons)
    Insect cell lines and baculoviruses, which do not infect human cells, allow bypassing many of the problems of glycoprotein overexpression. Despite being quite far from humans on the evolutionary tree, many fundamental biological processes, including proteins’ glycosylation, work very well in insects. Insect cell lines (ICL), derived from the butterflies fall armyworm (Spodoptera frugiperda) or Cabbage looper (Trichoplusia ni) in conjunction with baculovirus expression systems allow production of many glycoproteins in a serum-free medium.

    Insect cell lines

    The most commonly used insect cell lines, Sf9 and Sf21 from the fall armyworm, are used to amplify the recombinant virus. An additional advantage of the new generation insect cell lines such as cabbage looper’s High Five™ from Invitrogen is that they grow without CO2 at room temperature, as a semi-adherent monolayer or suspension – it’s much easier to work with, to start a facility from scratch (3). In fact, it’s been proposed to use large scale mammalian cell culture facilities or establish new facilities for vaccines production using insect cell lines (4).

    The vaccine against glycoprotein E of Japanese encephalitis virus has been produced in insect cells and has biochemical and biophysical properties equivalent to the mosquito antigen and causes antibodies formation (5). The other candidates for the insect cells produced vaccines include a vaccine against the main antigen determinants of the famous rabies virus, glycoprotein G.

    The use of insect cell lines for vaccine production is not limited to the glycoproteins, five commercially available vaccines for humans and animals, including CERVARIX® and and PROVENGE® for cervical and prostate cancer, respectively, are already in use and at least 8 others are in different stages of drug development (4).
    If your goals are more ambitious than protein purification and functional analysis, check out this blog article with tips about preparing glycoproteins for crystallization.

    2013-06-04-fall_armyworm_protein_production
    References:

    1. Ghaderi, D., Zhang, M., Hurtado-Ziola, N., & Varki, A. (2012). Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation Biotechnology and Genetic Engineering Reviews, 28 (1), 147-176 DOI: 10.5661/bger-28-147
    2. Vanoni, O., Paganetti, P., & Molinari, M. (2008). Consequences of Individual N-glycan Deletions and of Proteasomal Inhibition on Secretion of Active BACE Molecular Biology of the Cell, 19 (10), 4086-4098 DOI: 10.1091/mbc.E08-05-0459
    3. Working with insect cells (Invitrogen manual)
    4. Cox, M. (2012). Recombinant protein vaccines produced in insect cells Vaccine, 30 (10), 1759-1766 DOI:10.1016/j.vaccine.2012.01.016
    5. Kuwahara M, & Konishi E (2010). Evaluation of extracellular subviral particles of dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera frugiperda cells for use as vaccine and diagnostic antigens. Clinical and vaccine immunology : CVI, 17 (10), 1560-6 PMID: 20668137