Webinar Q&A Regarding Nanoparticle Albumin Bound Technology

Microfluidics’ recent webinar entitled “Comparison of Biotest HSA and Recombinant HSA for the Preparation of Nano Albumin Bound (nab) Paclitaxel using Microfluidizer® Technology” was well engaged and received a great deal of interest.

Our esteemed presenters, Senior Scientist Dr. Carolin Tetyczka at RCPE and Biotest’s Global Product Manager Dr. Liye Maeyama were joined with Microfluidics’ application engineer, Matt Laferriere, to discuss the processability and properties of nab-Paclitaxel using Biotest HSA in comparison to a commercially available recombinant HSA. In addition, quality parameters including content of small peptides, fatty acids, aggregates, binding characteristics of site II and the redox state in terms of cysteine-34 of Biotest HSA and other commercially available HSA solutions were discussed.

At the end of the webinar, the speakers were asked a great number of specific questions. Due to the overwhelming interest in this subject matter, our speakers regathered to provide further insight. Below is a list of the questions with abbreviated responses. The following video is a compilation of the questions asked along with a more detailed explanation. A timestamp is provided by each question to locate the full answer on the video.

(Please note: Answers may be reworded from the video for better clarification.)

The Impact of Microfluidizer^® Technology in NAB Production

1. Is Microfluidizer® technology able to be sterile filtered? (1:33)
   Yes, Microfluidizer® processers can be sterile filtered as long as the formulation reaches a sterile filtration of 0.22 micron.
2. Does Microfluidics have any exclusivity for Paclitaxel-Albumin product manufacturing? (2:00)
   No, Microfluidics International Corporation does not have any exclusivity.
3. Specific to the study and Microfluidizer® technology, what’s the batch size? (2:25) For this study, the batch size is 30 mL. For Microfluidizer® processors the batch size can range from 1 mL to thousands of liters depending on the application.  
4. What is the typical number of cycles used to obtain a 100-130 nm nanoparticle size? (2:50)
   Six cycles were used for this study at 1,200 bar to obtain nanoparticles in the range of 130 nm.

The Role of Solvents in Nanoparticle Albumin Bound Technology

1. Which chamber and what solvent was used for this experiment? (3:30)
   For this experiment a G10Z chamber was chosen, and a mixture of chloroform and ethanol was used as the organic solvent.
2. Was solvent removed prior to the high-pressure processing and which is the best method to remove chloroform – by TFF (tangential flow filtration) or rota- evaporation (rotary evaporator)? Is solvent simply distilled? Any aggregations? (4:03)
   For this study, the solvent was not removed prior to the processing. Neither TFF nor a rotary evaporator was used, and no aggregations were observed after the process.
3. What is the concentration of chloroform or organic solvent in the final product? (5:25)
   Residual solvent values were not investigated because it was not part of this project, however, the residual solvent limit for chloroform is 50 ppm and according to the assessment report for Abraxane^® there were tests performed but no data was presented.
4. What is the effect of dielectric constant of solvent used on particle size? What is the effect of aqueous soluble solvent on particle size with Biotest HSA? (6:14) Ultra-pure water was used to dilute the nanoparticles and according to the specifications of the Litesizer, which was used to investigate particle size and zeta potential, the optimum concentration was investigated in preliminary experiments. Thereby, it was found that 1:1 dilution with water and nanoparticles was optimal to reach particle sizes and zeta potential values in the appropriate range. The percentage of ethanol in the solvent was very low and the solvent mixture did not have a large impact on the active ingredient.
5. Is pre-saturation with solvent required for Biotest HSA and if so, why? (7:16)
   It is required. Since the study wanted to compare the results obtained with the market product of Abraxane, the patented process was used as a basis for the experiment and followed.

Study Specifics of RCPE

1. Does albumin concentration affect the final size of the NP? (7:58)
   Yes, it does. Higher concentrations of albumin lead to larger particle sizes.
2. How is pre-emulsification performed? (8:23)
   The organic phase consisted of the drug paclitaxel, dissolved in a mixture of chloroform and ethanol; this phase was added to the HSA-included aqueous phase before high-speed mixing via Ultra-Turrax for 5 minutes at 12,000 rpm.
3. Which was the efficiency of conversion of HSA to HSA-NP? Was the residual monomeric/polymeric HSA measured? (9:22)
   Regarding the particle size obtained, only one particle size fraction was obtained with a very narrow particle size distribution. No monomeric/polymeric HSA was detected. The encapsulation efficiency of paclitaxel in the nanoparticles was about 98%.
4. To prepare HSA-NPs to approximately 100-130 nm, which HSA concentration is typically used? (10:07)
   The applied HSA concentration was 1%.
5. Are the HSA-NPs stored in solution or as solid/lyophilized material? (10:20) Although the marketed product is lyophilized, for this experiment, the material was characterized and then removed.
6. Was there an attempt to sterilize through 0.22 filtration? What kind of membrane material was used in this study? What was the filtration yield? (10:47)
   No, filtration studies were performed for this study. So called Amicon^® filters separated the particles from the aqueous phase, which was subsequently used for the encapsulation efficiency studies.

Biotest Contributions in NAB Production

1. Further explain the importance of Cys 34 in Albumin? (11:50)
   The only free thiolgroup of Cys 34 in the albumin molecule is contributing to antioxidant property which represents approximately 80% of the antioxidative capacity of plasma. Cys 34 in its reduced state neutralizes free radicals, reactive oxygen species and reactive nitrogen. The degree of oxidized cysteine (Cys) 34 in HSA correlates with oxidative stress related diseases. It seems that the redox state of HSA influences the production of nab-paclitaxel, but this field needs more exploration.
2. Is Biotest HSA for manufacturing use suitable for injectables? What is the percentage of Biotest HSA used/needed as excipients for a CAR-T therapy? For example, in Kymriah what would be the estimated percentage? (13:20)
   Yes, Biotest can be used for manufacturing injectable finished products. It is not a medicine (from the regulatory point of view) but has the same manufacturing process and quality as the medicine according to the EP and is batch released by the Paul Ehrlich Institute in Germany. In Kymriah, the HSA concentration is 5% Human Serum Albumin (HSA).
3. Why is it a good point to have a lower fatty acids content for stem cells? (15:22)
   Lower fatty acid concentration means that the Human Serum Albumin is purer- meaning it is more chemically defined. It supports the reproducibility of stem cell culture. Fatty acids have an influence depending on the stem cells and their differentiation stage.  Palmitate, for example, causes endoplasmic reticulum stress and apoptosis in human mesenchymal stem cell and cell media containing reduced fatty acids bound to albumin has a positive impact on the growth of Murine embryos.
4. What are the other main ingredients in Biotest HSA? Stabilizers? (16:54)
   Sodium caprylate and N-acetyltryptophanate are used as stabilizers. 
5. What is the albumin stability with respect to pH and what is the suitable pH adjusting agent? (18:18)
   Biotest HSA has the pH between 6.7 – 7.3 according to the requirements of Pharm EUR (physiological pH). Investigations on the pH stability of HSA show, that HSA in general, is stable at pH 4-11 (there is slight variances depending on the HSA isomers), some 4-11, one isomer's optimum would be 7-10.
6. Is Bio test HSA a finished product and can it be used as an excipient? (19:29) Yes, it can be used as an excipient. Biotest HSA has the same manufacturing process as the medicine, and it can be used as an excipient in a finished product to be injected. Biotest HSA itself has no drug license but has the pharmaceutical quality. 
7. Is HSA recombinant or plasma extracted? (20:05)
   HSA is not recombinant and is plasma derived.
8. Is Biotest excipient grade HSA sold in USA and is it US FDA certified? (20:44) Biotest HSA containing cell media is sold in the US.  The cell media are being used in cell therapy products. Biotest HSA has no FDA drug license. 
9. Please explain the in vivo process and how was the encapsulation efficiency measured? (22:28)
   In-vivo, nab-paclitaxel undergo a dynamic dissolution process into smaller nanoparticles, albumin-bound paclitaxel complexes and unbound paclitaxel. The main involved transport of nab-paclitaxel into tissues and tumors occurs via active transcytosis mediated by albumin receptors and caveolin-mediated transport.
   For the encapsulation efficiency, commercially available Amicon^® centrifugal filters were used to separate the nanoparticles from the aqueous phase. The amount of paclitaxel was then quantified in the aqueous phase via HPLC.

If you should have further questions, please reach out to our presenters directly:

• Dr. Carolin Tetyczka at RCPE (Research Center Pharmaceutical Engineering)
• Dr. Liye Maeyama at Biotest
• Matt Laferriere at Microfluidics International Corp.

Watch our entire webinar to learn more.