The Guideline on Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related products and the informal chapters
of the pharmacopeia e.g. USP <1787> and USP <1790> suggest the monitoring and investigation of aggregates by methods that are orthogonal to the pharmacopeia methods. The following figure provides an overview ofhow the orthogonal methods cover different size ranges as well as different levels of information obtained. We adhere to highquality standards and procedures and have been successfully audited by the US FDA and numerous customers.
In addition to the compendial method mentioned in USP <788>,USP <787> and the EUROPEAN PHARMACOPOEIA (2.9.19. PARTICULATE CONTAMINATION), we at rap.ID have also implemented methods orthogonal to light obscuration to complete the investigation on sub- visible and visible
Light obscuration is the only compendial method ranging from the clearly subvisible (1 µm) up to 400 microns using the HCP-LD-50/50 sensor for the Pamas SVSS. The maximum particle concentration is up to 24,000 particles per milliliter. At this concentration the unit goes into coincidence and therefore two particles could pass the sensor at the same time. The analysis volume ranges from
100µl up to 1,000 ml. Low volume methods are also compatible with USP <787>. This makes this unit ideal for the application of USP <788> measurement of sub-visible particulate matter , using the small volumes of sample that are typically available in biopharmaceutical drug development. The instrument is calibrated with spherical particles, therefore only the sizes of spheres are true values. All other shapes are re-calculated as a sphere by the instrument. No morphological information is provided.
The sampling mechanism is similar to Light Obscuration as the sample is pumped through a detector. Rather than passing through a light source, a high-magnification camera collects images of the particle and morphology is obtained. Assumptions can be made that spheres are Silicone droplets and that irregularly shaped particles are of a proteinaceous nature. The smallest sample size is also 100 microliters. The ideal size range is 2-10 micrometers. Different levels of magnification make it possible to measure particulates up to 2000 µm.
The Single Particle Explorer, SPE ls raman.ID instrument created by rap.ID covers a size range from 1 µm up to 5 mm particles. By using the in situ Raman wet-dispersion.AID device, the instrument provides shape and morphologic information on particles and aggregates in the same manner as the FlowCam. Additional micro Raman spectroscopy delivers the chemical structure of the molecules forming protein particles. Even the with mass resonance measurement(RMM) , previously undetectable mixtures of Silicone oil and Protein particles can be identified making this the ultimate orthogonal method to the USP <788>; Light Obscuration. Enumeration is performed by means of static image analysis, thus a 20X Objective is able to count and size as well as morphologically determine particles larger than 2 µm.
Utilizing this new wet cell design (wet-dispersion.AID) we
challenge the SPE raman.ID and demonstrate its ability to count, size and identify visible and sub visible particles in the protein based therapeutic (in situ). Simultaneously, isolation of protein aggregates with the traditional filtr.AID device is also possible and is extremely efficient for the identification of visible and subvisible particles isolated from larger volumes.
We were able to transfer the visible particle into the novel wet-dispersion.AID without any changes in the particle’s appearance. Within only 20x Raman spectroscopy shows
that the particle consists mainly of the protein API with a content of about 10-20% Silicone. Leading to the conclusion that this aggregation was induced by Silicone droplets.
In our FDA registered and inspected contract testing laboratory we have established the compendial Light Obscuration (USP <788>) as well as FlowCam measurements for the enumeration of subvisible particles.
With the novel wet-dispersion.AID the SPE instrument uses micro Raman to identify hundreds of Silicone droplets and Protein API agglomerates in a Protein based therapeutic formulation. Preliminary testing show great promise for control and stability development of biopharmaceutical formulations (inherent and instrinsic particulate matter) usingin situraman.ID to optimize exhipients and minimize inherent protein aggregates. In the chapter Parenterals we cover the application of the SPE instrument on foreign particulate matter (extrinsic).
Our novel wet-dispersion.AID coupled with the powerful features of the SPE raman.ID instrument can provide a unique insight to protein interactions in your formulations.
The SPE enables you to conduct silicone protein inter-action studies or other formulation stability related investigations. Measuring and identifying particles is now as easy as a touch of a button.
Laskina, O., Lee, K. A., Lankers, M., Valet, O. (2016) Image Directed identification of Sub-Visible Particles in Protein Based Therapeutics. Classification According USP<787> of Intrinsic, Inherent and Extrinsic Particulate Matter on the Sub-Visible Level, Pittcon.
Laskina, O., Lankers, M., Valet, O. (2016) Methods to Help Control and Distinguish “Inherent”, “Extrinsic”, and “Intrinsic” Particulate Matter in Pre-Filled Syringes by Membrane Microscopy with Image Directed Raman Spectroscopy, 2016 Pre-Filled Syringes Forum.
Laskina, O., Valet, O., Lankers, M. (2016) Protein Aggregation Control in Parenterals, PEGS The Essential Protein Engineering Summit.
Laskina, O., Valet, O., Lankers, M. (2016) Siliconized Syringes: Analysis of the Quality and Stability Testing, AAPS National Biotechnology Conference.
Laskina, O., Valet, O., Lankers, M. (2016), Application of In Situ Raman Spectroscopy to Support Root Cause Investigation of Particulate Matter in Parenterals, AAPS National Biotechnology Conference.
Laskina, O., Valet, O., Lankers, M. (2016) Connecting MFI Shape Information with Raman Spectroscopic Intelligence for Analysis of Proteins, Silicone and Fatty Acids in Liquid Formulations and on Filters, AAPS National Biotechnology Conference.
FDA USP 788. (2009). Guidance for Industry Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the Guidance for Industry. Evaluation, (December). Retrieved from http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm085366.pdf
LANKERS, M., O. VALET, (2008), Differentiation between foreign particulate matter and silicone oil induced protein aggregation in drug solutions by automated Raman spectroscopy, Microscopy and Microanalysis, 14 (Suppl. 2), Conference 2008
VALET, O., LANKERS, M., (2008), Higher Yield and Quality through Particle Identification, Journal of the IEST, October 2008
DAS, T., (2007), Early Stage Protein Formulation Development and Use of High Throughput Screening Methods, AAPS NBC , San Diego 2007
LANKERS, M., (2004), Differentiation between Foreign and Protein Particles in Biopharmaceutical Preparations , RISBM 2004 "Raman and IR Spectroscopy in Biology and Medicine"