Name: failure of cleaning verification in pharmaceutical industry due to uncleanliness of stainless steel surface
Uploaded: 2017-08-11T15:00:00
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1. Sample Solution
<ol>
	<li>Calculate the cleaning limit (CL) for a drug based on the maximum allowable carry-over (MACO) according to the therapeutic dose criteria. 
	NOTE: Here, the cleaning limit (CL) for drug A has been calculated to be 2.4 &#181;g per 50 cm2 based on the maximum allowable carry-over (MACO) according to the therapeutic dose criteria. The cleaning verification is executed at the 50%, 100%, and 150% cleaning limit. The cleaning verification has been assessed for two formulations (2.5...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Validation of cleaning processes (7/93). Guide to inspections validation of cleaning processes. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 320-12-08, Gulf Pharmaceuticals. , (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 320-12-058, Novartis Internationsl AG. , (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 05-10, Teva Parenterals Medicines, Inc. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 320-14-09, Tianjin Zhongan Pharmaceutical Co., Ltd. , (2014).</li><li>Mantel, M., Wightman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+surface+chemistry+on+the+wettability+of+stainless+steel.">Influence of the surface chemistry on the wettability of stainless steel.</a> Surf Interface Anal. 21, 595-605 (1994).</li><li>Odaka, K., Ueda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+outgassing+rate+on+surface+oxide+layer+thickness+in+type+304+stainless+steel+before+and+after+surface+oxidation+in+air.">Dependence of outgassing rate on surface oxide layer thickness in type 304 stainless steel before and after surface oxidation in air.</a> Vacuum. 47, 689-692 (1996).</li><li>Kerber, S. J., Tverberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stainless+Steel+Surface+Analysis.">Stainless Steel Surface Analysis.</a> Adv. Mater. Processes. , 33-36 (2000).</li><li>Kusumaningrum, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+foodborne+pathogens+on+stainless+steel+surfaces+and+cross-contamination+to+foods.">Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods.</a> Int J Food Microbiol. 85, 227-236 (2003).</li><li>Liu, L., Pack, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+verification+assays+for+highly+potent+compounds+by+high+performance+liquid+chromatography+mass+spectrometry:+Strategy,+validation,+and+long-term+performance.">Cleaning verification assays for highly potent compounds by high performance liquid chromatography mass spectrometry: Strategy, validation, and long-term performance.</a> J Pharmaceut. Biomed. 43, 1206-1212 (2007).</li><li>Lambropoulos, J., Spanos, G. A., Lazaridis, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+HPLC+assay+for+fentanyl,+alfentanil,+and+sufentanil+in+swab+samples.">Development and validation of an HPLC assay for fentanyl, alfentanil, and sufentanil in swab samples.</a> J Pharmaceut. Biomed. 23, 421-428 (2000).</li><li>Chisti, Y., Moo-Young, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clean-in-place+systems+for+industrial+bioreactors:+Design,+validation+and+operation.">Clean-in-place systems for industrial bioreactors: Design, validation and operation.</a> J. Ind. Microbiol. 13, 201-207 (1994).</li><li>Haidar Ahmad, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+verification:+Exploring+the+effect+of+the+cleanliness+of+stainless+steel+surface+on+sample+recovery.">Cleaning verification: Exploring the effect of the cleanliness of stainless steel surface on sample recovery.</a> J Pharmaceut. Biomed. 134, 108-115 (2017).</li></ol>

The major contributor to low and variable recoveries of API residues from stainless steel coupons was traced to the lack of a well-defined procedure for cleaning of the coupon surfaces. Cleaning the surface of the coupons resulted in consistent, accurate spiked recovery and reproducible results. With the demonstration of high recoveries from stainless steel coupons, the actual cleaning verification results obtained from the manufacturing equipment using validated method(s) should be accurate and precise, reflective of th...

The cleanliness of non-dedicated equipment should be verified before its subsequent release for use in the manufacture of intermediates and active pharmaceutical ingredient (APIs), at product change over to prevent cross-contamination. Cleaning procedures should contain sufficient details to enable operators to clean each type of equipment in a reproducible and effective manner, and these procedures should be validated according to the U.S. Food and Drug Administration (FDA) requirements1. Numerous warning letters due to inadequate cleaning2,3,4, failure to validate the cleaning verification method, and failure to follow cleaning procedures5 have been issued by the FDA. 21 CFR &#167;211.67 outlines the requirements needed for successful cleaning verification.
It is the standard in industry that the validation of the analytical methods for cleaning verification is performed on stainless steel coupons with the same surface/finish as the manufacturing equipment. Stainless steel coupons (e.g., 50 cm2) are used to represent equipment surfaces for cleaning verification experiments in the laboratory. During the development and validation of these analytical methods, the sample of interest (i.e., the residues that should be recovered from the surface of the equipment) is spiked at the target residue onto the stainless coupon determined by the maximum allowable carry-over (MACO) limit. This level is determined based on acceptable exposure limit which is defined as the limit at which a patient can get exposed with no adverse health effects (no-observed-adverse-effect-level, NOAEL).
The analyst or manufacturing operator conducting the swabbing must follow a structured procedure to ensure that recoveries are reproducible regardless of who performs the swabbing. The procedure should explicitly detail the swab type, number of swabs used, the diluent, the amount of solvent used, the exact sweeping pattern, the number of strokes applied to the sampling surface, the amount of time spent swabbing/extracting the samples, the method of detection (ultra-violet, fluorescence, mass spectrometry, total organic carbon, etc.), the extraction technique of the material from the swab head, etc.
Besides all the above-mentioned factors that affect sample recovery, the surface of the coupon, and thus, the surface of the equipment also play a role. The surface of the coupon may be modified due to the deposition of a thin film of material on the surface or due to change in the oxidation state of one or more of the elements in stainless steel (e.g., Fe, Cr, and Ni)6,7,8. The regeneration of the surface of the stainless steel coupons back to its original state is vital for the success of the quantitative swapping process. Studies, in which the stainless steel coupons were not properly cleaned, showed variability in recovery including analyst to another, different drugs, or various spike levels9,10,11. The standard deviation in recovery of ten replicates on one coupon can be up to 14% and 26% on five coupons9. It is important to note that the relative standard deviation (Srel) values increased with increasing number of replicates or with increasing the number of coupons used (i.e., five coupons instead of spiking five times on the same coupon)11. In such cases, the variability cannot be interpreted as random fluctuation. Nonetheless, their results can be explained by our finding that the cleanliness of the surface of the coupon will affect the recovery. The results described in this paper illustrate a significant increase in recovery results and decrease in variability after properly cleaning the surface of the stainless steel coupons.
Clean-in-place (CIP) is an automated way of cleaning the surface of equipment that involves a minimal or no disassembling of the equipment. During the CIP cleaning process, a defined procedure of consecutive wash with a base followed by an acid is executed to remove organic and inorganic residues. Surfactants, chelating compounds, or complexing agents are usually added to the CIP solutions to enhance the efficiency of cleaning any product from the surface of the equipment. The efficiency of cleaning depends on several parameters including the choice and concentration of the CIP solutions (i.e., type and composition of base, acid, and surfactant), the cleaning time, temperature (typically 60 - 80 &#176;C), contamination type, and the presence of hard to clean parts12. Based on the type of drug product, CIP solutions 100 and 200 have been chosen to use for cleaning the stainless steel coupons used for cleaning verification, since it simulates the CIP process used for cleaning the manufacturing equipment.
This study reports the influence of different factors affecting the recovery of pharmaceutical residues from the surface of stainless steel coupons and recommends the best practices for analytical cleaning method development for small molecules, therapeutic proteins, and antibodies. The lack of a well-defined procedure that consistently cleaned coupon surface was identified as the major contributor to low and variable recoveries. High and reproducible recovery was obtained when the surface of the coupon was cleaned properly13.

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		<col style="width:401pt" width="535" />
		<col style="width:250pt" width="333" />
		<col style="width:48pt" width="64" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="width:265pt;height:15.75pt" width="353">stainless steel coupons</td>
			<td class="xl67" style="width:401pt" width="535">&#160;GlobePharma (New Brunswick, NJ ).&#160;</td>
			<td class="xl16" style="width:250pt" width="333">SS316-20RA-50cm2</td>
			<td style="width:48pt" width="64"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl16" height="21" style="height:15.75pt">Clean in place solutions (CIP100 and CIP200)&#160;</td>
			<td class="xl68" style="width:401pt" width="535">were obtained from Steris Corporation (Mentor, OH)</td>
			<td>1D10BG</td>
			<td>Alkaline detergent and acid detergent, respectively</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl16" height="21" style="height:15.75pt">Positive displacement pipettes</td>
			<td class="xl68" style="width:401pt" width="535">Gilson (Middleton, WI).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="34" style="height:25.5pt">
			<td height="34" style="height:25.5pt">HPLC grade water</td>
			<td class="xl68" style="width:401pt" width="535">Millipore Milli-Q Advantage Water Purification System (Darmstadt, Germany)&#160; or from Honeywell Burdick &#38; Jackson (Muskegon, Michigan)</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl16" height="21" style="height:15.75pt">HPLC grade Methanol</td>
			<td class="xl68" style="width:401pt" width="535">EMD</td>
			<td>MX0475-1</td>
			<td></td>
		</tr>
		<tr height="17" style="height:12.75pt">
			<td height="17" style="height:12.75pt">glacial acetic acid&#160;</td>
			<td class="xl68" style="width:401pt" width="535">EMD</td>
			<td>MAX0073P5</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl16" height="21" style="height:15.75pt">HPLC grade Acetonitrile&#160;</td>
			<td class="xl68" style="width:401pt" width="535">J.T. Baker (Avantor Performance Materials, Center Valley, PA)</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="17" style="height:12.75pt">
			<td height="17" style="height:12.75pt">Trifluoroacetic acid</td>
			<td class="xl68" style="width:401pt" width="535">J.T. Baker (Avantor Performance Materials, Center Valley, PA)</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="height:15.75pt">Chromatography column Zorbax Eclipse</td>
			<td class="xl68" style="width:401pt" width="535">&#160;XDB-C18, 4.6 x 100 mm, 3.5 &#181;m HPLC column</td>
			<td>UNSPSC &#8211; 41115709</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl16" height="21" style="height:15.75pt">Vanquish UHPLC system&#160;</td>
			<td class="xl68" style="width:401pt" width="535">Thermo Fisher Scientific, Germering, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl16" height="21" style="height:15.75pt">Branson B8510 Ultrasonic cleaner&#160;</td>
			<td class="xl68" style="width:401pt" width="535">Branson Ultrasonics (Danbury, CT, USA)</td>
			<td>model (8510-D7H)</td>
			<td></td>
		</tr>
	</tbody>
</table>

failure of cleaning verification in pharmaceutical industry due to uncleanliness of stainless steel surface

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A series of factors were assessed, including drug product spike levels, spiking procedure, drug-excipient ratios, analyst-to-analyst variability, intraday variability, and cleaning procedure of the coupons. The lack of a well-defined procedure that consistently cleaned the coupon surface was identified as the major contributor to low and variable recoveries. Assessment of cleaning the surface of the coupons with clean-in-place solutions (CIP) gave high recovery (&#62;90%) and reproducible results (Srel&#8804;4%) regardless of the conditions that were assessed previously. The approach was successfully applied for cleaning verification of small molecules (MW &#60;1,000 Da) as well as large biomolecules (MW up to 50,000 Da).

Representative results from the initial attempts for cleaning verification for drug A are summarized in Table 2. Before the coupons were cleaned according to the detailed procedure in the experimental section, inconsistent results were obtained at different spike levels, for various ratios of API/excipient, different analysts, and even for the same analyst at different days. The observed variability in the recoveries needed to be addressed, as some of the results failed t...

Watch this Scientific Journal Video about Failure of Cleaning Verification in Pharmaceutical Industry Due to Uncleanliness of Stainless Steel Surface at JoVE.com

Failure of Cleaning Verification in Pharmaceutical Industry Due to Uncleanliness of Stainless Steel Surface

The lack of a well-defined procedure that consistently cleaned coupon surfaces was identified as the major contributor to low and variable recoveries in cleaning verification. This manuscript describes the correct protocol of cleaning stainless steel coupons.

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A ...

The overall goal of this work is to obtain high and reproducible recoveries of pharmaceutical residues from the surface of stainless steel coupons by applying the best practices for analytical cleaning method development for small molecules, therapeutic proteins and antibodies. This method can help answer key questions in the field of cleaning verification in the pharmaceutical industry, such as why cleaning verification of manufacturing equipment fails. The main advantage of this study is that it shows the procedure to properly clean the surface of the stainless steel coupons used for analytical method validation.To start this experiment, prepare sample solutions of the drug dissolved in the diluent of choice after calculating the cleaning limit for a drug. Clean the stainless steel coupons by rinsing with water and wiping the surface twice for 10 to 15 seconds. Then, repeat this rinsing and wiping step with methanol to eliminate any residual deposit.To perform the advanced cleaning approach using clean in place solutions, immerse the coupons in high performance liquid chromatography or HPLC grade water and sonicate for two minutes. Immerse the coupons in 0.1%alkaline detergent solution in HPLC grade water and sonicate for two minutes. Immerse the coupons in HPLC water again and repeat two minute sonication.Then, immerse them in 0.1%acid detergent solution in HPLC water and sonicate for two minutes. Finish with two minute sonication in HPLC water. Next, using a double-sided tape, mount the stainless steel coupons on the bottom of a 250 milliliter plastic beaker.Hold the beaker during the procedure to facilitate spiking and swabbing and to avoid accidents. Use a positive displacement pipette to infuse a defined drug volume, at a desired concentration and formulation, in a whirly pattern on the coupon surface. Once the coupon surface is dry, dip a dry swab in a vial with two milliliters of diluent.Press the swab against the inside of the vial to remove excess solvent. Use even, overlapping side-to-side strokes to firmly wipe the coupon surface with one side of the wet swab, until the total 50 square centimeter test area is wiped. Use the same side of the swab to repeat the wiping process.Swab each of the four edges of the coupon twice. Rotate the coupon 90 degrees and use the other side of the swab, now rotating the direction of wiping by 90 degrees. Repeat the cleaning using this side of the swab in the same fashion, making sure to wipe all the four edges of the coupon twice.After the surface wiping, use scissors to cut the swab head into the solvent vial. After that, repeat the whole swabbing procedure using a second swab, while rotating the coupon 90 degrees toward the same direction chosen before. Next, sonicate the vial containing two swab heads for five minutes.Then, vortex the vial for 10 seconds. Finally, transfer the solution into an HPLC vial and label it as the working solution. To recover the sample, prepare the control solutions by mixing 200 microliters of each coupon spiking or sample solution with 1, 800 microliters of diluent.Use these solutions to run chromatography according to the text protocol. Calculate the recovery of the swab working solution based on the relative area under peaks of the working and control solutions. Repeat this process for three coupons to calculate the average recovery and the relative standard deviation.Before the coupons have been cleaned, recovery results for different spike levels were inconsistent, regardless of active pharmaceutical ingredient or API excipient ratio, different analysts, or even different day for the same analyst. The majority of the recovery results had high relative standard deviation. The observed variability in the recovery was not eliminated when several different parameters were adjusted, including swabbing technique, the diluent, the spiking solvent, the pH of the spike and the diluent, the spiking technique and the extraction technique.The difference between the individual recovery at each coupon surface and the average recovery shows that the average recovery is different from one coupon surface to another, therefore, it is very likely that the observed variability comes from the differences between the coupon surfaces. After following the coupon cleaning described in this protocol, the recovery results were reproducible from one trial to another, with minimal difference in recovery between the coupons. The coupon cleaning performed here resulted in the recoveries being higher than 90%Furthermore, relative standard deviation values for each spiking level were acceptable and smaller than the results obtained before the cleaning.Once mastered, the cleaning of the coupons can be done in less than 30 minutes, if performed properly. While attempting this procedure, it's important to remember how to spike and recover properly without losing the analyze of interest. After watching this video, you should have a good understanding of how properly clean stainless steel surfaces to obtain high and reproducible recoveries of the residues.After its development, this technique paved the way for researchers in the pharmaceutical industry to provide reliable cleaning data for manufacturing processes.

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Chemistry

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

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We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

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This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

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Here, we present a one-pot, transition-metal-free synthesis of thiols and thioesters from aromatic halides and sodium thiomethoxide, followed by the preparation of single crystals of a metal-dithiolene network using thiol species generated in situ from the more stable and tractable thioester.

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and ...

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Herein, detailed protocols for the oxidative iodination of terminal alkynes using hypervalent-iodine reagents are presented, which chemoselectively afford 1-iodoalkynes, 1,2-diiodoalkenes, and 1,1,2-triiodoalkenes.

We present the chemoselective synthesis of 1-(iodoethynyl)-4-methylbenzene, 1-(1,2-diiodovinyl)-4-methylbenzene, and ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Surface Properties of Synthesized Nanoporous Carbon and Silica Matrices

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) with a 4.6 nm pore size, and ordered silica porous matrix, SBA-15, with a 5.3 nm pore size. This work describes the surface properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the differently ordered porous materials with similar pore sizes. For this purpose, OMC and SBA-15 with highly ordered nanoporous structures are synthesized via impregnation of the silica matrix by applying a carbon precursor and by the sol-gel method, respectively. The porous structure of investigated systems is characterized by an N2 adsorption-desorption analysis at 77 K. To determine the electrochemical character of the surface of synthesized materials, potentiometric titration measurements are conducted; the obtained results for OMC shows a significant pHpzc shift toward the higher values of pH, relative to SBA-15. This suggests that investigated OMC has surface properties related to oxygen-based functional groups. To describe the surface properties of the materials, the contact angles of liquids penetrating the studied porous beds are also determined. The capillary rise method has confirmed the increased wettability of the silica walls relative to the carbon walls and an influence of the pore roughness on the fluid/wall interactions, which is much more pronounced for silica than for carbon mesopores. We have also studied the melting behavior of D2O confined in OMC and SBA-15 by applying the dielectric method. The results show that the depression of the melting temperature of D2O in the pores of OMC is about 15 K higher relative to the depression of the melting temperature in SBA-15 pores with a comparable 5 nm size. This is caused by the influence of adsorbate/adsorbent interactions of the studied matrices.

Here we report the synthesis and characterization of ordered nanoporous carbon (with a 4.6 nm pore size) and SBA-15 (with a 5.3 nm pore size). The work describes the surface and textural properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the materials.

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &amp;#945;-Imino &amp;#947;-Lactones and Alkylidene Pyrazolones

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN&#8216;s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple ...