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LNP Formulation Process Capability Expansion in Caponago

Edition 1 / March 2022
Lipid & Injectable Platforms > New Lipid NanoParticle Formulation Capabilities Bring Flexible Drug Development Options

Edition 1 / March 2022

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Umberto Romeo, R&D Manager, CordenPharma Caponago, headshot

Author
Umberto Romeo,

R&D Manager
CordenPharma Caponago


Close up of girl smiling at Pediatrician holding oral solid dose pills in their hand

New Lipid NanoParticle Formulation Capabilities Bring Flexible Drug Development Options

Colorful rendering of Lipid NanoParticle (LNP) with its payload.

CordenPharma Lipid NanoParticle with its payload.

The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of Lipid NanoParticle (LNP) delivery systems.

LNPs are liposome-like structures especially geared towards encapsulating a broad variety of genetic payloads, primarily nucleic acids (DNA-RNA based) including siRNA, microRNA, mRNA and saRNA. In late 2020, CordenPharma initiated a strategic investment program at the Sterile Injectable facility CordenPharma Caponago (IT) to build out a new LNP formulation process capability, as well as to have in-house analytical techniques to control the finalized LNP-assembled product. The investment was designed to be fully-integrated with the already-existing Fill & Finish clinical & commercial capacity in place.

The 2-Phase Investment Includes:

  • >> Phase 1 – To build up, via expansion of an existing approved R&D Laboratory, an LNP formulation technology area with a target to conduct LNP familiarization, as well as small / lab-scale development of any LNP formulated drug. This phase was completed in January 2022.
  • >> Phase 2 – Via a different, but significant, plant expansion for an already-existing GMP area to build up an LNP manufacturing suite for the production of LNP-formulated batches from small to large-scale. Anticipated completion of this phase is early 2023.

Focusing on the LNP R&D Laboratory that is already operational, we introduced an array of technologies to support LNP-based formulation development services, including Lipid NanoParticle analytical characterization and quality control.

There are two main methods for producing Lipid NanoParticles: top-down and bottom-up processes. Top-down approaches, such as thin-film hydration and ethanol injection, are commonly named bulk methods. They often need non-standard multi-step procedures to reduce the particles’ size and polydispersity. These batch methods are usually coupled with size-reduction steps such as sonication or extrusion.

To overcome these drawbacks, some continuous production techniques, such as Impingement Jet mixing and microfluidics, have been developed and are now available not only at bench-scale but also in a cGMP setting. With these methods, identified as a bottom-up approaches through rapid micro-mixing techniques, (L)NP are self-assembled, without requiring complex multi-step manufacturing processes. Once the (L)NP assembly process is complete, the formulation requires an Ultrafiltration / Diafiltration (UF/DF) step.

At CordenPharma Caponago we introduced the following technologies for (L)NP assembly, based on bottom-up approaches:

  • >> An R&D unit Impingement Jet mixing by Knauer: Encapsulation is achieved through the collision of two streams at high velocity in a jet-mixing chamber. One of the streams contains the lipids in organic solvents and the other the genetic payload in acidic buffer. The mixing at high velocity reduces the solubility of the lipids through a swift change in polarity so that homogenous nanoparticles are formed. The quality of nanoparticles can be controlled through the streams’ flow stability, the mixer geometry and the fluid velocities. The R&D Skid contains one mixing unit constituted by three pumps generating a Total flow up to 0.1mL/min. Through parallelization, the same technology is available at larger-scale supporting clinical and commercial cGMP manufacture. This same technology is used for the commercial supply of Covid-19 mRNA-LNP based vaccines.
  • >> A Microfluidic Automated Lipid Nanoparticle System (ANPS) by Dolomite: This equipment exploits chip-based microfluidic technology. Chips are available with different geometries to enable micro-mixing including Microfluidic Hydrodinamic Focusing (MHF) Chips, in which lipid nanostructures are formed at the stream interface due to an aqueous solution and ethanol (containing the lipids) diffusion, resulting in diffusive mixing and local dilution of the organic phase. This technology can be used for screening purposes working on volumes ranging from micro-scale (200µL) up to several mL.
  • >> A NextGen Microfluidic Blaze Plus system by Precision Nanosystems (PNI): This equipment is based on a proprietary mixing technology evolved from the Staggered Herringbone Mixer (SHM) using a chaotic advection micro-mixer in which the mixture phenomenon, where the advected particles repeatedly transverse vortices, leads the fluids to wrap into one another (commonly called the “stretching and folding” process). This technology was created to overcome the productivity limitation of MHF, allowing the production of lipid nanostructures at higher flow rates and improving the mixture efficiency. This technology can process volumes from 10mL to 10L. The same technology is available also at cGMP scale.

Lab tech wearing blue gloves operating microfluidic equipment for R&D at CordenPharma Caponago.

Microfluidic equipment for early / small-scale R&D activities in CordenPharma Caponago (IT).

Additionally, the following systems have been introduced to support UD/DF process development steps:

  • >> ÄKTA™ flux s and ÄKTA™ flux 6 by Cytiva: They are cross-flow filtration systems for sample concentration and diafiltration (buffer exchange). The system allows filtrations using natively hollow fiber filters, cartridges, cassettes, and membrane absorbers. The ÄKTA flux s can be used for research and filter screenings or to process small volumes, while the ÄKTA flux 6 for process development and small-scale production. The combined systems offer the possibility to exploit a filtration surface from 0.2cm2 to 1.2m2.
  • >> Cogent µ and Cogent M1 by Merck: the Cogent μScale tangential flow filtration (TFF) system is an easy-to-use, semi-automated benchtop solution that has been designed to fully support TFF process development at the “micro-scale” using up to three 88 cm2 cassettes (264 cm2). It has a low minimum working volume (16 mL*), the ability to operate at feed pressures up to 80 psi (5.5 bar), and low pulsation (≤ 3 psi). The Cogent M1 is a benchtop TFF system with automatic TMP control suited for process development and scaling studies and small-scale production. It is equipped with a 10-liter tank with embedded balance, and it can run in fed-batch or diafiltration mode using an optional transfer pump, which enables the system to process up to 100 liters based on product and membrane filter characteristics. According to flow characteristics, the system can run from 0.1m2 to 0.5m2 filter membrane cassettes.

Caponago technician reviews ultrafiltration equipment for concentration of LNP-formulated drugs.

Equipment for ultrafiltration / concentration of LNP-formulated drugs at CordenPharma Caponago (IT).

In addition, new analytical technologies have been acquired in order to enable nanoparticle characterization, analytical method development and quality control to ensure product quality including:

  • >> Dynamic Light Scattering (DLS) coupled with MALS (Multi Angle Light Scattering) technology by Malvern, enabling not only particle-size distribution measurements, but also the determination of sample concentration and Z Potential measurements.
  • >> Microplate reader by BioTek to perform RNA assay testing through RiboGreenTM assays or similar kits capable to quantify as low as 1ng of RNA.
  • >> Agarose Gel Electrophoresis (AGE) coupled with an iBrightTM by Invitrogen detection system for nucleic acids identification/quantitation.
  • >> (H)UPLC Dual Chromatographic system equipped with DAD/DAD/CAD detection by Thermofisher for lipid quali-quantitative analysis.

Caponago operators collaborate in the R&D laboratory for LNP formulation & analytical testing.

The R&D Laboratory at CordenPharma Caponago (IT) with expanded services for LNP formulation and analytical testing.

Finally, CordenPharma Caponago relies on a network of partners to support us with additional testing for physical / biological nanoparticles characterization (e.g., Cryo TEM and/or in vitro testing) of the manufactured LNP-based formulations, as well as a global partner for mRNA payloads.


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