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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).
    New biotech company giving private-sector boost to SA drug development research

    New biotech company giving private-sector boost to SA drug development research


    SolubleBioScience has yet to move its headquarters to San Antonio, but it’s already having a direct impact on advancing drug development in the Alamo City. The Birmingham, Alabama-based biotech company, recently acquired by CytoBioscience, is contributing new technology to the Center for Innovative Drug Discovery that could prove to be a game changer for researchers and patients.

    SolubleBioScience, a new wholly owned subsidiary of CytoBioscience, has developed an instrument called the HSC, which uses proprietary technology to combine high throughput analytical capabilities with a predictive algorithm to optimize protein formulations.

    The gesture by CytoBioscience and SolubleBioScience is part of a larger effort by the two companies to bring more private-sector support to the Center for Innovative Drug Discovery – or CIDD – and to San Antonio’s bioscience sector.

    UT Health President Dr. William Henrich said the HSC instrument, the first of its kind in the world, will expedite San Antonio scientists’ efforts to move research findings from the laboratory to discovery and ultimately to patient treatments. The instrument, which costs $225,000 each, represents a significant advancement in science, shortening the formulation process from a full year to less than two months.

    James Garvin – CEO of CytoBioscience, which moved its headquarters from Germany to San Antonio in 2015 – said the HSC will improve the accuracy and expediency of research work at the CIDD.

    I previously reported that CytoBioscience plans to move SolubleBioScience from Birmingham to San Antonio before the end of the year. That’s a key reason why UT Health and UTSA are the first academic institutions in the world to get SolubleBioScience’s HSC technology.

    “One of the things I believe with all my heart is that we have to be good corporate citizens and we have to work with our community partners,” Garvin said. “UT Health is one of our best partners. So, to me, this was a no brainer. This is great technology. We wanted to work with one of our partners who could really use it and see what we can do together.”

    Bruce Nicholson, founder of the CIDD, said the gift will go to the center’s High Throughput/Content Screening facility on UT Health’s campus.

    “It’s the second gift we have received from CytoBioscience,” Nicholson said. “What this new instrument will do is allow high throughput testing of solubilities. Many drugs need to be in high concentrations to be effective, but you can only get them to limited concentrations in a lab setting.”

    Nicholson is particularly impressed with the fact that help is coming from a pair of companies still getting their bearings in San Antonio.

    “It’s truly unprecedented,” he said “Normally, academic institutions are not going to get access to this [technology]. But I think CytoBioscience’s whole concept is that the best way to advance drug discovery is to build instruments that have industrial applications but to also share them with academia, which can fuel the enterprise and continue to generate discoveries. So this is very generous of them. But it’s also forward thinking.”

    Matt Hart, director of the CIDD’s high throughput screening facility, said the HSC will add a new dimension to the center’s capabilities.

    “This further emphasizes the importance of academic and industry collaboration,” he said. “This type of collaboration is going to be key in growing the bioscience industry in San Antonio.”

    Former German firm bringing more biotech business to San Antonio

    Former German firm bringing more biotech business to San Antonio

    San Antonio’s successful recruitment of Cytocentrics more than a year ago has led to another economic win that could further enhance the city’s role in the global bioscience arena.

    The company, which recently changed its name to CytoBioScience Inc., has acquired a Birmingham, Alabama-based biotech firm called Soluble Therapeutics that will look to establish roots here over the next 12 months.

    CytoBioScience officials believe the “multimillion-dollar purchase” could draw more companies and talent to the Alamo City. At a minimum, it will likely attract more attention from entities that could be looking for a new home.

    “What this does is deepen the biotech footprint in San Antonio. And whenever you do that, you just don’t know what shore the ripples from these waves will land on,” CytoBioScience CEO Dr. James Garvin told me.

    CytoBioScience, which decided to move its headquarters from Germany to San Antonio in 2015, develops and manufactures devices that allow other researchers, medical institutions and pharmaceutical companies to better understand how human cells react to medicine. The company’s acquisition of Soluble Therapeutics is part of a larger strategic shift aimed at broadening its product platform.

    Soluble Therapeutics Managing Director Sharney Logan said the acquisition will allow CytoBioScience to expand its technology to more global markets. As part of the agreement, the Alabama biotech firm will change its name to SolubleBioScience and move its operations, including its scientific team, to San Antonio.

    Garvin expects other bioscience companies will take note of San Antonio’s latest win.

    “This will continue to raise the awareness of what is already happening in the city,” he said.

    CytoBioScience leadership hinted recently that the company was looking to widen its reach. And the acquisition of Soluble may not be its last move.

    But the deal could trigger other economic opportunities as San Antonio continues to build up its bioscience industry assets, as well as its reputation across the United States and abroad.

    “San Antonio is already one of the best places in the world for biotechnology,” Garvin said. “But we need to continue to beat our chests.”

    Soluble Therapeutics Acquired by CytoBioscience

    Soluble Therapeutics Acquired by CytoBioscience

    SAN ANTONIO–(BUSINESS WIRE)–CytoBioscience, Inc. has purchased Birmingham, Alabama-based Soluble Therapeutics and will move the company’s operations to its San Antonio location.

    The deal closed November 1, according to James Garvin, Chief Executive Officer of CytoBioscience. Details of the multi-million dollar purchase price were not disclosed.

    “The Soluble Therapeutics team has created a business offering that aligns very well with our technology and work to positively impact the efforts of drug development enterprises and researchers across the globe,” he said. “This deepens our product offering in the market and also strengthens our scientific team. We look forward to integrating their team and delivering an expanded set of capabilities to the biotechnology and pharmaceutical industries.”

    “Combining forces with CytoBioscience will dramatically increase our business, as it will provide additional service opportunities to our existing customers,” said Sharney Logan, Managing Director of Soluble Therapeutics. “Additionally, this transaction will provide new opportunities to expand our technology in global markets. As part of the acquisition, Soluble Therapeutics will also be changing its name to Soluble BioScience to reflect its increased capacities.”

    Soluble employs 8 people and the plans are for the company to move to San Antonio within the next year.

    About Soluble Therapeutics, Inc.

    Soluble Therapeutics, Inc (now Soluble BioScience) was founded in 2008 to commercialize the HSC™ Technology which is licensed from the University of Alabama at Birmingham. The company’s services enhance the drug development process by rapidly optimizing protein solubility and stability.

    About CytoBioscience, Inc.

    CytoBioscience creates and manufactures devices that represent a convergence of bioscience, nano science, microchip development, algorithms and engineering. The company’s devices allow other researchers, medical institutions and pharmaceutical companies to better understand how human cells react to medicine. CytoBioscience was founded in Germany and moved its headquarters and manufacturing to San Antonio in 2015.

    Contacts
    for CytoBioscience, Inc.
    Jim Dublin, 210-387-3113
    jdublin@dublinstrategies.com