Engineering Conferences International

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Fougerolles, Tony De

ENGINEERING EXOSOMES TO ENABLE AND CREATE IMPROVED GENETIC MEDICINES 
 
Tony De Fougerolles, Evox Therapeutics Limited, United Kingdom 


English

Engineering Exosomes to Enable and Create Improved Genetic Medicines

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Khvorova, Anastasia

LIPIDATED CONJUGATES FOR CNS DELIVERY 
 
Anastasia Khvorova, University of Massachusetts, USA 
 


Lipidated conjugates for CNS delivery

Hermans, Guy

ENDOSCAPE: TRITERPENE GLYCOSIDE CONJUGATES ALLOW EFFICIENTLY ENDOSOMAL ESCAPE 
OF DIVERSE TARGETED PAYLOADS 
 
Dr. Guy Hermans, CEO, Sapreme Technologies B.V., The Netherlands 
hermans@sapreme-technologies.com 
 
 
Triterpene glycosides are secondary plant metabolites, some of which co-evolved with ribosomal inactivating 
proteins to allow their efficient entry into the cytosol. Building on two decades of academic research into their 
biology, Sapreme and its partners have constructed and validated the ENDOSCAPE technology platform which 
allow for cell type specific cytoplasmic delivery of a variety of payloads. Here, we will present data on a range of 
in vitro and in vivo experiments functionally demonstrating targeted delivery of oligonucleotides and proteins to 
the cytoplasm. 
 
 


ENDOSCAPE: Triterpene glycoside conjugates allow efficient endosomal escape of diverse targeted payloads

Badr, Sara

Gupta, Priyanka

Workshop - Sustainability (Training Workshop), sponsored by EVOTEC

Sinclair, Andrew

Hill, Claire

Abe, Yuki

Ojeilli, Sa’ad

ECO-DESIGN: ALTERNATIVES TO PROCESS MASS INTENSITY IN ASSESSING THE IMPACT OF 
BIOPROCESSING 
 
Andrew Sinclair, Biopharm Services Ltd, 
 a.sinclair@biopharmservices.com  
Claire Hill, ProPharma Group 
Dr. Yuki Abe,. Biopharm Services Ltd 
Sa’ad Ojeilli, Biopharm Services Ltd 
. 
Key Words: Bioprocessing, sustainability ecodesign 
Process Mass Intensity (PMI) is commonly used as a proxy for environmental impact during process 
development. In contrast to process development, manufacturing facilities are assessed on CO2e emissions to 
measure their environmental impact. The value of PMI needs to be determined, and alternative measures for 
use in process development that better align with the metrics used by operating facilities need to be identified. 
This study evaluates the value of PMI and its relationship to Greenhouse Gas Warming Potential (GWP) as 
measured by Scope 2 CO2 equivalents g per g of product produced. The analysis is based on mAB 
manufacturing runs at different capacities, comparing Stainless Steel, Single-Use, and several process 
intensification options. 
This study used the BioSolve Process.1 (BSP) to model manufacturing operations by mapping scaleless 
process recipes linked to databases of commercially available industry-benchmarked equipment, consumables, 
and materials. It calculates a facility's mass and energy balance. Key outputs are production schedules, 
bottleneck analysis, Bill of Materials, and equipment requirements, and these are used to predict the cost of 
goods, capital, water use, PMI, and energy use for both process and building. The model includes all the critical 
functions of the manufacturing asset (labor, solution management, QC, utilities, etc). In this study, The focus is 
on sustainability metrics: PMI, including both process and cleaning accounting for water, plastics, and materials 
(material efficiency); Process Intensity, the amount of product manufactured per unit volume of manufacturing 
space per year g/m3/yr (facility efficiency), Energy Intensity accounting for process and building (Boiler & 
Electrical) KWh/g and operating Operating CO2 Equivalence taking into account the level of carbonization of 
electrical supply and the boiler fuel. The building energy metrics rely on location metrology data provided in the 
model to account for the historical annual weather conditions.  
The first step was to compare the model's predictions with published data. Our primary source is the Budzinski 
paper.2 This references actual PMI data and LCA data. The model's predicted PMI for a Single-Use facility is 
within 10% of the published data across the main PMI categories (8,766 vs. 9092). For the CO2e/g, there is a 
closer match with the published data: 22.54 CO2e g/g vs. BPS 22.17 CO2e g/g. 
Although PMI is commonly used in process development as 
a sustainability guideline, the study demonstrates the lack of 
insight into energy usage from facility operations, which 
means this metric is not aligned with at least one of the 
critical drivers of facility sustainability: carbon emissions. A 
common misconception about PMI is that it is scale-
independent. While this applies to small molecules, we have 
found that PMI is not scale-independent for biologics, 
especially if cleaning is involved in SS processes. The ratio 
between water, materials, and consumables categories is 
also scale-dependent for SU processes. 
While a full LCA is the most accurate environmental impact 
assessment, it is not practical as an Eco-Design tool in early development. While no single measure can 
accurately represent all aspects of sustainability, carbon emissions are a focus for many organizations’ 
sustainability efforts due to their broad-ranging impact on human health. Predicting carbon emissions in a 
simplified format, such as combining PMI with Process Intensity, could provide guidance better aligned with 
current facility metrics (GWP). The study includes an assessment of the impact of process intensification on 
both PMI and alternative metrics. When incorporated into the software, these metrics offer an alternative metric 
to PMI that better aligns with organizations' sustainability initiatives. 
                                                          
1 BSP is commercially available and a defacto standard across the biopharm supply chain with over 600 users 
2 1Budzinski, Kristi, et al. “Streamlined Life Cycle Assessment of Single Use Technologies in Biopharmaceutical 
Manufacture.” New Biotechnology, vol. 68, May 2022, pp. 28–36, https://doi.org/10.1016/j.nbt.2022.01.002.  
 


Eco-Design: Alternatives to Process Mass Intensity in Assessing the Impact Of Bioprocessing

Portez, Bea

Noonan,, Alexander

Mullerpatan, Akshat

Wu, Yang

Keegan, Jesse

Roper, Mark

SCALING UP CONTINUOUS BIOPROCESSING: INSIGHTS FROM SMALL-SCALE PRODUCTION 
 
Bea Portez, Sanofi 
Bea.Portez@sanofi.com 
Alexander Noonan, Sanofi  
Mark Roper, Sanofi  
Jesse Keegan, Sanofi  
Yang Wu, Sanofi 
Akshat Mullerpatan, Sanofi 
 
 
Key Words: Perfusion, Continuous Bioprocessing, CHO, Scale-up  
 
Pushing the boundaries of continuous bioprocessing in large scale operations remains to be an emerging area 
of interest in the biopharmaceutical industry. Numerous notable progressions in facilitating technologies for 
integrated and continuous bioprocessing have surfaced over recent years. The end-to-end process in Sanofi 
looks to incorporate an intensified perfusion production process to demonstrate the end-to-end process 
integration with downstream purification steps to produce a drug substance material. Through a single batch 
run, we developed a perfusion model capable of supporting two months of intensified production using a new 
cell line. We plan to test different molecules in multiple demonstrational experiments with the goal of achieving 
successful implementation in GMP. 
 
In this study, a scale down model was conducted using four, 3L benchtop bioreactors in perfusion mode as a 
risk mitigation strategy for a continuous end-to-end 500L pilot-scale perfusion production of a monoclonal 
antibody (mAb) by CHO cells. The 3L benchtop reactors, operating in duplicate, utilized two different processes, 
maintaining a steady state of ≥ 100 M viable/cells for a duration of two months. Monitoring key indicators and 
metabolites such as lactate dehydrogenase, sieving, glucose, lactate, osmolality, etc., was crucial in ensuring 
the success of the long perfusion process. Our findings facilitated the determination and implementation of an 
effective scale-up process to a 500L perfusion pilot-scale, achieving productivity that supported the end-to-end 
process. This case study also proves that process optimization in small-scale bioreactors continuous to be 
essential for identifying and addressing issues to prevent setbacks and failures during large-scale production.  
 
 
 
  


Scaling Up Continuous Bioprocessing: Insights from Small-Scale Production

Andersson, Andreas

Maude, Henrik

Castan, Andreas

Ross, Neil

Romann, Patrick

SOLVING THE SEPARATION CHALLENGE IN PERFUSION: LAB-SCALE VERIFICATION AND PILOT-
SCALE IMPLEMENTATION 
 
Andreas Andersson, Cytiva, Sweden 
andreas.andersson@cytiva.com 
Henrik Maude, Cytiva, Sweden 
Neil Ross, Cytiva, Scotland 
Patrick Romann, Levitronix, Switzerland 
Andreas Castan, Cytiva, Sweden 
 
 
Key Words: perfusion, TFF, sieving, Starling recirculation, filtration performance, ccTFF 
 
Tangential flow filtration (TFF) systems using hollow fibers are the primary choice for scalable cell retention in 
perfusion processes. However, product retention emerges as a critical challenge, affecting both yield and 
process longevity. The product retention can be described by the sieving coefficient which is calculated as the 
ratio between product concentration in the harvest and in the bioreactor. This is impacted both by the filter 
properties such as pore size, lumen ID and path length, and the operating parameters of the system, including 
shear rate, permeate flux and the trans membrane pressure (TMP) profile. 
 
A constant TMP is often assumed in TFF, however practical observations reveal a dynamic TMP profile with 
high TMP in the initial fiber segments and negative TMP at the exit. This leads to a backflow phenomena known 
as Starling flow. Recent literature1 proposes a way to mitigate Starling flow by establishing a filtrate pressure 
gradient using a pump to create a co-current filtrate flow, described as co-current TFF (ccTFF). 
 
This study implements a two-pump ccTFF system in both laboratory-scale and pilot-scale perfusion processes. 
The results demonstrate the maintenance of a high sieving coefficient over 18 days and a substantial 
improvement over standard TFF. Importantly, ccTFF implementation did not adversely affect cell culture 
performance or product quality, ensuring consistent downstream product concentrations. Furthermore, this 
study illustrates how the simple modification of existing TFF systems to support ccTFF operations can 
accommodate bioreactor volumes between 28 and 500 L and enhance steady-state operations. 
In summary, the implementation of ccTFF presents a promising solution to enhance product passage in 
perfusion processes to improve process efficiency and downstream predictability. This research contributes 
valuable insights into the optimization of bioprocess technology for sustainable and high-yield production. 
 
 
 
Reference: 
1 Romann, P et. al (2024). Co‐current filtrate flow in TFF perfusion processes: Decoupling transmembrane 
pressure from crossflow to improve product sieving. Biotechnology and Bioengineering,121, 640–
654.https://doi.org/10.1002/bit.28589654 
 
  


Solving the separation challenge in perfusion: Lab-scale verification and pilot scale implementation

Mehta, Ushma

Implementation of Raman Spectroscopy for in-line monitoring of critical process parameters of CHO cell 
perfusion cultures 
 
Ushma Mehta, MilliporeSigma, USA 


Implementation of Raman Spectroscopy for in-line monitoring of critical process parameters of CHO cell perfusion cultures

Kruse, Thomas

Kampmann, Markus

Schmitz, Fabian

THE RABBIT JUMPS AHEAD – RAPID A BIOSMB BIOLAYER INTERFEROMETRY TECHNOLOGY 
 
Markus Kampmann, Sartorius, Corporate Research 
Markus.Kampmann@Sartorius.com 
Thomas Kruse, Sartorius, Corporate Research 
Fabian Schmitz, Sartorius, Corporate Research 
 
 
Key Words: continuous capture, membrane adsorber, multi-column chromatography, PAT 
 
Monoclonal antibodies (mAbs) are nowadays fundamental in treating a wide range of severe diseases, including 
cancer, infections, autoimmune disorders, and inflammatory diseases. Due to the high specificity, potent activity 
and fewer side effects compared to conventional drugs, the market for mAbs rises continuously. Ongoing 
improvements in the upstream process, such as higher mAb titers, have shifted the bottleneck in mAb 
production to the downstream process (DSP). Consequently, there is an increasing demand for DSP process 
intensification technologies to overcome manufacturing bottlenecks. Semi- and fully continuous processes are 
gaining traction in both industry and academia due to their cost-effectiveness and efficiency. However, the 
complexity of these processes, throughput limitations and the need for adaptive control strategies present 
significant hurdles.  
 
Continuous multi-column chromatography (MCC) offers several benefits, such as increased productivity, 
reduced buffer consumption, and lower production costs, all within a smaller equipment footprint. However, 
MCC processes commonly rely on predefined loading volumes and flow rates. This requires several 
assumptions like a constant flow rate and known mAb concentration of the feed solution, that all columns have 
the same dynamic binding capacity (DBC) and that the DBC remains constant throughout the process. These 
challenges could be overcome by a dynamic loading based on breakthrough detection of the mAb.  
This study introduces an enhancement of a recently developed MCC approach (RC-BioSMB, [1]) that leverages 
convective diffusive membrane adsorbers (Sartobind® Rapid A) by a dynamic loading approach based on 
at-line biolayer interferometry measurements [2]. This approach addresses the limitations of conventional UV 
spectroscopy for dynamic loading, such as low specificity and high background noise from media components, 
by detecting mAb breakthrough with greater specificity. A continuous production setup was established by 
incorporating a continuous virus inactivation and sterile filtration. 
 
Setpoint variations were successfully addressed by a dynamic flow control throughout all unit operations as well 
as a dynamic loading of the RC-BioSMB process. Despite the significantly steeper mAb breakthrough with 
membrane adsorbers compared to resins, an enhanced membrane utilization was obtained while 
simultaneously minimizing product losses. The convective diffusive membrane adsorbers enabled a high 
productivity of 697 g L-1 d-1 for the RC-BioSMB process. A robust continuous process was operated for several 
days. A high overall yield of 87 % with simultaneous high removal of process-related impurities like host cell 
proteins (3.3 log removal to 70 ppm) was obtained throughout the process. The results of this study and the 
novel employed approaches demonstrate substantial potential for the establishment of adaptive continuous 
manufacturing of biopharmaceuticals. In addition, the continuous at-line biolayer interferometry prototype 
enables the potential extension of this technology to the chromatographic purification of other biological 
modalities, such as recombinant proteins or viruses. 
 
 
[1] Schmitz, F., Knöchelmann, E., Kruse, T., Minceva, M., & Kampmann, M. (2024). Continuous multi-column 
capture of monoclonal antibodies with convective diffusive membrane adsorbers. Biotechnology and 
Bioengineering, 1–17. https://doi.org/10.1002/bit.28695 
[2] Kruse, T., Austerjost, J., Lemke, J., Krasov, Y., Popov, V., Pollard, D., & Kampmann, M. (2024). Advanced 
control strategies for continuous capture of monoclonal antibodies based upon biolayer 
interferometry. Biotechnology and Bioengineering, 121, 771–783. https://doi.org/10.1002/bit.28586 
 


The RABBIT jumps ahead - Rapid A BioSMB Biolayer Interferometry Technology

Kundu, Anita

Dhingra, Ashna

Coffman, Jon

Lee, Ken

Radle, Matt

Woodall, Elise

Squeri, Andrea

McGuin, Jana

Haggerty, Patrick

A FULLY AUTOMATED PERFUSION PROCESS USING RAMAN SPECTROSCOPY 
 
Anita Kundu, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
anita.kundu@astrazeneca.com 
Ashna Dhingra, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Patrick Haggerty, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Jana McGuin, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Elise Woodall, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Andrea Squeri, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Matt Radle, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Ken Lee, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
Jon Coffman, Biopharmaceutical Development, AstraZeneca Gaithersburg, USA 
 
 
Key Words: Raman spectroscopy, process controls, PAT, automation, process characterization 
 
Raman spectroscopy is a light scattering technology which produces spectra encoding the chemical fingerprint 
of a sample. These spectra can be paired with offline measurements to build a chemometric model to predict 
real-time metabolite data in the permeate stream of a perfusion bioreactor. Unlike traditional perfusion 
methodologies that aim to maintain a steady state, AstraZeneca’s integrated continuous manufacturing platform 
is dynamic which adds complexity to the control strategy. The current upstream continuous platform utilizes 
offline measurements and operator inputs to update the flowrates for a cell bleed and multiple concentrated 
feeds once daily. The presented automated control strategy uses real-time metabolite data collected by Raman 
spectroscopy and a proportional integral algorithm to control all pump flowrates at the 3L scale. This fully 
automated perfusion process eliminates the need for offline bioreactor samples and reduces manual data entry 
requirements, thereby reducing operator time requirements and improving process reproducibility. Scalability of 
the automated process is demonstrated at the pilot (50L) and clinical manufacturing (500L) scales without 
additional modifications to the 3L control strategy. Additionally, this bioreactor control strategy can be coupled 
with an automated aseptic sampling system and mass spectrometer to obtain real-time product quality results 
without manual sample manipulation. By synergizing the two automation strategies, process characterization 
can be fully automated, thus alleviating the significant resource requirements incurred during late-stage pipeline 
activities. 


A fully automated perfusion process using Raman spectroscopy

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