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\begin{document}

\title{Accelerating Attribute-Focused Process and Product Development Through
the Development and Deployment of Autonomous Process Analytical
Technology Platform System}



\author[1]{Chao-Hsiang Wu}%
\author[1]{Pik Chan}%
\author[1]{Zac Sarich}%
\author[1]{Yaokai Duan}%
\author[1]{Janice Chen}%
\author[1]{Jiu-Li Song}%
\author[1]{Mike Berke}%
\author[1]{Les Miranda}%
\author[1]{Chetan Goudar}%
\affil[1]{Amgen Inc}%


\vspace{-1em}



  
  \date{April 28, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Enabling real-time monitoring and control of the biomanufacturing
processes through product quality insights continues to be an area of
focus in the biopharmaceutical industry. The goal is to manufacture
products with the desired quality attributes. To realize this rigorous
attribute-focused Quality by Design (QbD) approach, it's critical to
support the development of processes that consistently deliver
high-quality products and facilitate product commercialization. Time
delays associated with off-line analytical testing can limit the speed
of process development. Thus, developing and deploying analytical
technology is necessary to accelerate process development. In this
study, we have developed the Micro Sequential Injection (\selectlanguage{greek}µ\selectlanguage{english}SI) process
analyzer and the Automatic Assay Preparation Platform (A2P2) system.
These innovations address the unmet need for an automatic, online,
real-time sample acquisition and preparation platform system for
in-process monitoring, control, and release of biopharmaceuticals. These
systems can also be deployed in laboratory areas as an off-line
analytical system and on the manufacturing floor to enable rapid testing
and release of products manufactured in a GMP environment.%
\end{abstract}%



\sloppy


\textbf{Accelerating Attribute-Focused Process and Product Development
Through the Development and Deployment of Autonomous Process Analytical
Technology Platform System}

Chao-Hsiang (Richard) Wu, Becky Chan, Zac Sarich, Yaokai Duan, Janice
Chen, Jiu-Li Song, Mike Berke, Les P. Miranda, and Chetan T. Goudar

Attribute Sciences, Process Development

Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320

Corresponding Author:

Chao-Hsiang (Richard) Wu

chaow@amgen.com

805-279-6740

The authors declare no conflict of interests.

\textbf{Short Running Title:}

\textbf{Autonomous PAT Systems in Accelerated Product Development}



\textbf{ABSTRACT}

Enabling real-time monitoring and control of the biomanufacturing
processes through product quality insights continues to be an area of
focus in the biopharmaceutical industry. The goal is to manufacture
products with the desired quality attributes. To realize this rigorous
attribute-focused Quality by Design (QbD) approach, it's critical to
support the development of processes that consistently deliver
high-quality products and facilitate product commercialization. Time
delays associated with off-line analytical testing can limit the speed
of process development. Thus, developing and deploying analytical
technology is necessary to accelerate process development. In this
study, we have developed the Micro Sequential Injection (\selectlanguage{greek}µ\selectlanguage{english}SI) process
analyzer and the Automatic Assay Preparation Platform (A2P2) system.
These innovations address the unmet need for an automatic, online,
real-time sample acquisition and preparation platform system for
in-process monitoring, control, and release of biopharmaceuticals. These
systems can also be deployed in laboratory areas as an off-line
analytical system and on the manufacturing floor to enable rapid testing
and release of products manufactured in a GMP environment.

\textbf{KEYWORDS}

Process Analytical Technology (PAT), Quality by Design (QbD), Micro
Sequential Injection (\selectlanguage{greek}µ\selectlanguage{english}SI), Automatic Assay Preparation Platform (A2P2)

\textbf{INTRODUCTION}

The biomanufacturing landscape has transformed recently, impacting labor
demand, causing supply chain disruptions, and leading to persistent
inflation. Workforce adaptations have led to productivity disruption and
increased health concerns for those unable to work remotely. To stay
competitive, innovation is crucial to adapt to the new norm and to
introduce new products. Expediting the introduction of new products
requires an understanding of critical product quality attributes,
safety, efficacy, manufacturing, and logistics. Reliable data is
essential for rapid development and speedy delivery to patients. As
remote work is now part of business continuity, proactive data
collection and analysis are needed for faster insights and value
creation. An innovative analytical strategy is vital for building
resilience and managing risks in manufacturing quality, addressing labor
demand, and securing a competitive edge.

Analytical testing is frequently the critical path in the entire product
manufacturing life cycle, often causing delays in product release due to
long testing result turnaround times. In next-generation process
development and manufacturing, real-time or near real-time analytics are
crucial to streamline QbD development through assay automation, data
visualization, and predictive modeling. This approach enables
attribute-focused development, robust manufacturing processes, and
rapid, yet reliable, process control and product release.

In developing an analytical strategy, automated systems aim to enable
routine and on-demand acquisition of key product quality and process
performance attributes in laboratories and manufacturing facilities. The
proposed autonomous PAT platform offers 24/7 monitoring, reliable
performance, and resource agility, as opposed to traditional analytical
approaches which require significant analyst intervention. Carefully
designed fluidic conduits and strategically placed material handling
mechanisms provide resource conservation, optimized assay efficiency,
controlled conditions, reproducible results, and long-term system
robustness. The system contains multiple functional unit operations,
working together through programmable algorithms to manage sample and
reagent logistics, direct processes, and reactions, and enable
self-directed assay protocol selection with rule-based parameter
adjustment for optimal output.

Drawing inspiration from next-generation manufacturing and the Industry
4.0 concept, analytical testing is being moved from laboratories to the
manufacturing floor. This paradigm shift enables in-situ testing for
faster results, improved process understanding, and greater
cost-effectiveness in process development and manufacturing {[}Ref.
1{]}. Sample acquisition and preparation workflows are typically prone
to errors due to manual operations. Meanwhile, liquid handlers require
substantial initial investment, extensive development, and can be costly
to adapt to new requirements. Chip-based fluidic miniaturization serves
as another option, offering advantages such as low material consumption,
design freedom, and rapid analysis {[}Ref. 2{]}.

Portability is another advantage of chip-based analytical systems,
allowing the co-location of the manufacturing process and analytical
instrument. This eliminates delays related to sample logistics and
laboratory testing. However, minimizing sample and reagent consumption
to the nanoliter or lower range on a microfabricated scale can introduce
sampling errors, analysis errors through evaporation, and assay errors
due to the precipitation of solids.

Instead, designing a compact and robust instrument that reduces sample
and reagent consumption to a few microliters is more advantageous. These
factors have led to a practical level of miniaturization through the
integrated \selectlanguage{greek}µ\selectlanguage{english}SI fluidic design. This design maintains short fluidic
conduit geometry instead of narrow ones, decreasing sample and reagent
consumption without the inherent issues of microfabricated fluidic
channels {[}Ref. 3{]}.

\selectlanguage{greek}\textbf{The µSI Methodology}\selectlanguage{english}

The \selectlanguage{greek}µ\selectlanguage{english}SI methodology, the successor to the flow injection (FI)
methodology, was introduced by Ruzicka and Marshall et al. {[}Ref. 4{]}.
It introduces computer control capability to the system, allowing for
better integration with modern process automation. A barebones \selectlanguage{greek}µ\selectlanguage{english}SI
system comprises a bi-directional syringe pump, a holding coil, a
multi-position valve, a reactor coil, and a detector. Conventional FI
protocols require physical reconfiguration of the flow manifold to
perform different assays; however, a \selectlanguage{greek}µ\selectlanguage{english}SI system allows all experimental
protocols to be scripted and selected by users.

In a reagent-based \selectlanguage{greek}µ\selectlanguage{english}SI assay protocol, the sample and the reagent are
injected sequentially through the solvent selector valve into the
holding coil, forming a layered solution profile in the tubing. The
solution profile is then transported through the mixing coil and into
the detector by the flow reversal action of the syringe pump after the
valve switches to the flow cell. A flow-through detector records the
changes in a desired physical parameter when the reaction product
reaches the flow cell.

Flow programming, along with random access to sample and reagent
solution ports by the valve, provides versatility since all operating
protocols such as sample injection, solute dispersion, and timing are
controlled by software protocols. Assays with similar characteristics
can be implemented on the same \selectlanguage{greek}µ\selectlanguage{english}SI system without physical
reconfiguration of the \selectlanguage{greek}µ\selectlanguage{english}SI system.

\selectlanguage{greek}\textbf{µSI Process Analyzer System}\selectlanguage{english}

The biomanufacturing industry has long aspired to achieve real-time
monitoring and control of biological manufacturing processes to
efficiently produce final products with desired product quality
attribute (PQA) profiles. With the aim of developing a system capable of
automatic end-to-end sample preparation, and to interface online/at-line
sample sources with analytical instruments to achieve multi-fold
acceleration in acquiring process performance and product quality
insights, the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system was designed and engineered.
The \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system comprises two compartments: the upper
sample processing compartment and the lower power supply compartment.
The sample processing compartment consists of four quadrants equipped
with syringe pumps with glass syringes, solvent selection valves, heated
coils, a degasser, solenoid valves, and a reagent cooler, all integrated
to minimize the fluidic path. This compact design reduces the footprint
and enables on-the-floor technology implementation.

\selectlanguage{greek}\textbf{Multi-Attribute Assay Sample Preparation in the µSI Process
Analyzer System}\selectlanguage{english}

The multi-attribute method (MAM) assay utilizes peptide mapping
methodology to identify and quantify critical quality attributes (CQAs).
It is typically performed off-line through manual sample handling,
followed by Liquid Chromatography-Mass Spectroscopy (LC-MS) operation,
and generating results can take several days. Transitioning the
off-line, manual MAM assay into a real-time online LC-MS integrated \selectlanguage{greek}µ\selectlanguage{english}SI
process analyzer system, where results can be obtained within 2 to 3
hours, can guide process development activities, and provide valuable
insights into the performance of a biomanufacturing process.

\selectlanguage{greek}\textbf{Glycosylation Profile Sample Preparation in the µSI Process
Analyzer System}\selectlanguage{english}

Monitoring the glycosylation profile is critical for biosimilar
products, where matching the glycosylation profile with the reference
product is essential to establish similarity {[}Ref. 5{]}. Off-line
glycosylation analysis involves manual bioreactor sampling followed by
the execution of the following three assays in tandem: (1) product
titer, (2) affinity purification, and (3) sample preparation and
analysis using Hydrophilic Interaction Chromatography (HILIC), resulting
in assay execution times of several days. In contrast, analysis times
for the on-line glycosylation assay using the HILIC-LC integrated \selectlanguage{greek}µ\selectlanguage{english}SI
process analyzer system are approximately 2 hours, providing rapid
feedback on product quality and process performance.

\selectlanguage{greek}\textbf{The Second Generation µSI Process Analyzer System: The A2P2
System}\selectlanguage{english}

To minimize the instrument's footprint, the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system
was integrated into a commercially available Agilent 1290 Infinity II
Ultra-High-Performance Liquid Chromatography (UHPLC) system and an
optional auxiliary mass spectrometer. This system enables both peptide
mapping and glycosylation assays and can function as a standalone sample
purification platform, complement the Tecan platform, or be used for
subsequent product quality assays like aggregation and charge variants.
This new system is referred to as the A2P2 system (\textbf{Figure 3} ).

Innovative designs enable user-selectable sample preparation protocols
to be executed on a platformed fluidic design, without physically
reconfiguring flow paths inside the A2P2 system. The design transforms a
typical UHPLC system into an A2P2-enabled LC system while maintaining
its original functionalities as a standalone LC system.

The A2P2 methodology enables the integration of modality agnostic
analytics with automatic sample acquisition, preparation, and analysis
in a compact form factor, resulting in a more than 50\% reduction in
footprint while providing enhanced functionalities. Additionally, the
A2P2 methodology can be used for online, at-line, or off-line
applications, enabling both on-the-floor and laboratory implementations.

\textbf{MATERIALS AND METHODS}

\selectlanguage{greek}\textbf{µSI Process Analyzer System for MAM Sample Prep}\selectlanguage{english}

The detailed \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer fluidic design for MAM sample
preparation is described in \textbf{Figure 1} . It consists of a series
of columns, reaction chambers, tubing, liquid redirect valves, a
multi-position solvent selector valve, and a syringe pump. Software
scripting capabilities enable the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system to
robustly execute the process steps described in \textbf{Table 1} .

Test samples can be acquired directly from the bioreactor supernatant or
purified material. The sample is aspirated into the system through the
solvent selector valve and passed through an affinity purification
column where the protein of interest is bound, and the sample matrix is
removed as column flow-through. The purified protein is then eluted and
delivered to a thermostatic incubator where denaturation, reduction, and
alkylation reactions occur. This chemically processed sample undergoes
buffer exchange and is enzymatically digested in a second thermostatic
incubator before chromatographic separation and mass detection.

\selectlanguage{greek}\textbf{µSI Process Analyzer System for Glycan Map Sample Prep}\selectlanguage{english}

In this implementation, N-linked oligosaccharides are enzymatically
released from samples using Peptide-N-Glycosidase F (PNGase F). The free
oligosaccharides are then derivatized with the fluorophore
2-aminobenzoic acid (2-AA) on the reducing terminal N-acetylglucosamine
(GlcNAc). The fluorescently labeled oligosaccharides are separated by
the HILIC analytical column and detected by fluorescence.
In\textbf{Figure 2} , the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system replaces manual
processes with automated sampling, sample purification and matrix
removal, enzymatic release of glycans, fluorescent labeling of glycans,
enrichment of labeled glycans, and synchronized injection, along with
chromatographic separation and quantitation of labeled glycans.

\textbf{Table 2} describes the glycan map sample preparation with
protein capture, sample matrix removal, and glycan release by on-column
enzymatic digestion in the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system. In\textbf{Figure
2a} , cell-free cell culture samples are acquired by the aspiration
action of syringe pump 1 and sent to the affinity purification column,
where the protein of interest is retained in the column, and the sample
matrix is removed as column flow-through. The enzyme is subsequently
introduced into the affinity column, and glycans are released in the
interstitial solution of the affinity column. This design eliminates the
need for protein precipitation. The released glycans are carried out by
syringe pump 1 and mixed with the glycan reagent from syringe pump 2
before the degasser. The degasser removes microbubbles formed when the
aqueous solution from the affinity column meets the organic solution of
the 2-AA reagent prepared in methanol. The degassed glycans and 2-AA
reagent mixture are delivered to the 80\selectlanguage{ngerman}°C reaction coil to accelerate
the fluorescent labeling reaction in flow-through mode. The 2-AA labeled
glycans are air-cooled and enriched by a porous graphitic carbon (PGC)
column while the excess 2-AA reagent and non-labeled glycans are cleared
as the PGC column flow-through. The enriched labeled glycans are
released from the PGC column and transferred to an LC injection valve,
where glycan quantitation is accomplished through HILIC chromatography.

\textbf{The Flow-Thru Vial (FTV) in the A2P2 system}

The FTV enables all sample processing functions in the A2P2 system and
directs processed intermediate and/or fully processed samples to return
from the sample preparation unit to the autosampler for further
processing and/or chromatographic injection. The FTV can also act as a
sample receptacle connected to the bioreactor or process stream,
enabling online sample processing. The design and fabrication of the FTV
were carried out in-house to ensure proper testing and quick turnaround
time for prototyping. As shown in \textbf{Figure 4} , a threaded hole
was used to connect the vial to the system, and an angled gutter at the
top of the vial allows liquid to flow directly to waste. As liquid flows
into the bottom of the vial, it forces excess liquid into the gutter,
where it can gravity drain to waste. Because the vial has unique
features, a vial holder was also fabricated in-house to ensure proper
vial alignment. The FTV was made from polyether ether ketone (PEEK) for
its chemical inertness, and the vial holder was made from aluminum.

\textbf{Process Execution in the A2P2 System}

The aim of creating the autonomous workflow in \textbf{Figure 5} is to
illustrate the A2P2 system's ability to conduct a lengthy laboratory
process autonomously while eliminating the need for an analyst's input:
titer measurement for the starting material, affinity purification for
the starting material, titer measurement for the purified sample, and
subsequent PQ analysis, in this case, the protein aggregation assay.
This workflow serves as a foundation for independent operation of all
applications documented in this paper, as well as for future A2P2 uses.
The System Suitability Testing (SST) and Intelligent Run Control (IRC)
functions are applied in concert, automating the process of sample
coordination, operating parameter generation, and transfer among the
A2P2 system actions. This substantially reduces human involvement,
mitigates transcription errors, enhances assay reliability, and
accelerates the decision-making process.

\textbf{RESULTS AND DISCUSSIONS}

\textbf{Real-time MAM Readout}

The LC-MS integrated \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system was introduced into a
manufacturing facility and connected to a process stream for continuous
product quality monitoring. The robustness of the system was first
verified by processing reference standard daily for 6 days, and the
results in \textbf{Figure 6a} were comparable to off-line analysis. The
system was then programmed for daily bioreactor sampling. As depicted
in\textbf{Figure 6b} , each bioreactor sample was bracketed by two
reference standards as an assay acceptance check, and product
deamidation and clips were measured. The staggering of sample processing
and LC-MS analyses resulted in a throughput of approximately 7 samples
per day.

\textbf{Real-time Glycosylation Data}

\textbf{Figure 7a} displays a typical glycan map generated using the
HILIC UPLC integrated \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system from preliminary
proof-of-concept experiments; the results were comparable to those
obtained off-line using the manual approach. Subsequently,\textbf{Figure
7b} verifies the integrated system's performance over a 32-day period,
using daily processing of the reference standard, indicating good system
repeatability and robustness. \textbf{Figure 7c}showcases results from
online measurements over a 32-day period from a production-scale
bioreactor. Each data point represents one bioreactor sample acquired
daily online, each sample is bracketed by two reference standards to
ensure assay acceptance and accuracy. This data provides insights that
can guide process development and aid in manufacturing consistent,
high-quality products.

\textbf{Bioreactor Product Titer Measurement by the A2P2 System}

In \textbf{Figure 8a} , biological samples, whether manually acquired or
automatically delivered, can be introduced into the autosampler via
regular sample vials or the FTV. The autosampler initiates sample
aspiration from these sources and carries out the injection into an
affinity chromatography column within the column compartment. A
spectrophotometric detector, such as a UV detector, then establishes the
product titer/concentration by comparing it with known concentration
standards.

\textbf{Bioreactor Product Aggregation Assessment by the A2P2 System}

Before protein aggregation assessment of any cell culture sample, an
analyst must perform affinity purification prior to injection into a
size exclusion (SEC) chromatography column. Matrix removal and protein
enrichment are essential before sample preparation and PQ assays.
Compared to the Tecan-Atoll process, the A2P2 system significantly
improves efficiency, reducing sample turnaround time by 30\%-64\%,
saving staff hands-on time by up to 92\%, and increasing sample
processing capacity by 90\%-154\%. \textbf{Figure 8a} depicts how the
autosampler needle takes either manually collected or online samples
from the vial or FTV and directs them through the affinity capture
column. The column captures the protein of interest, while the sample
matrix is flushed to waste through the FTV. The eluted protein of
interest accumulates in the FTV as the affinity-purified sample, which
can subsequently be injected into the analytical sizing column for
aggregation level quantification by a UV detector. Samples that are free
from cell culture matrix or have undergone affinity purification in
downstream processes require no further affinity capture/cleanup.
Analysts can directly inject these samples into the analytical sizing
column, and a UV detector quantifies their aggregation level.

\textbf{Bioreactor Product Charge Variant Profile Assessment by the A2P2
System}

To evaluate charge variant profile in a cell culture sample, the sample
must undergo affinity purification and buffer exchange to reduce the
salt content of the sample matrix before injection into an ion exchange
column. As shown in \textbf{Figure 8b} , the autosampler needle takes
manually collected or online samples from the vial or FTV and directs
them through an affinity capture column for cleanup. The column captures
the protein of interest, flushing the sample matrix to waste via the
FTV. The protein of interest is then eluted from the affinity capture
column and accumulates in the FTV as an affinity-purified sample. The
sample is subsequently diluted with the mobile phase of the charge
variant assay inside intermediate vials to lower the salt concentration
before injection into the analytical ion exchange column. If dilution
doesn't sufficiently reduce the sample's salt concentration, a desalting
column can perform a buffer exchange on the sample before injection for
analysis. However, samples that are free from cell culture medium and
meet the assay requirements for salt content require no affinity
capture/cleanup, dilution, or buffer exchange. These samples can be
directly injected into the ion exchange column for analysis.

\textbf{Bioreactor Product Glycosylation Profile Assessment by the A2P2
System}

In the case of sample preparation for a glycosylation profile assay, if
the starting material is a cell culture sample, the process includes
affinity protein cleanup, enzymatic release of glycans from the protein,
chemical labeling of the released glycans with fluorescent tags, and
subsequent HILIC chromatography column injection and analysis. As
depicted in \textbf{Figure 8c} , the autosampler receives manually
collected or automatically delivered bioreactor cell-free samples in
vials or through the FTV. The autosampler needle takes the sample and
injects it into the affinity capture column for cell culture matrix
removal. The autosampler needle then delivers PNGase F to the capture
column for on-column glycan release. The released glycans are
transferred to the FTV, mixed with 2-AA, and directed to the 80\selectlanguage{ngerman}°C
reaction coil, where the fluorescent labeling reaction takes place. The
fluorescently labeled glycans are then conveyed back to the FTV, ready
for normal-phase chromatographic separation and quantitation.

\textbf{Bioreactor Product Quality Assessment using MAM by the A2P2
System}

A cell-free cell culture sample must undergo affinity purification,
denaturation, reduction, alkylation, buffer exchange, and enzymatic
digestion before analysis. Occasionally, a secondary enzyme is required
to fully investigate product quality attributes. As shown
in\textbf{Figure 8d} , the autosampler receives manually collected or
automatically delivered bioreactor cell-free samples in vials or through
the FTV. The autosampler needle takes the sample and injects it into the
affinity capture column for cell culture matrix removal. The autosampler
then transports the purified sample into one of the intermediate vials
and sequentially adds denaturation and reduction reagents. Or, as an
alternative approach, the reagents can be directly added to the FTV to
streamline the process. The mixture is then delivered to and incubated
inside the 40°C reaction coil, in either the stopped-flow mode or the
preferred continuous-flow mode. After the protein is denatured and
reduced, it is transferred to the FTV, where the alkylation reagent is
added either directly or using a new intermediate vial. The mixture is
then directed through the 40°C reaction coil for incubation to complete
protein alkylation and is subsequently buffer exchanged using the
desalting column. The desalted, reduced, and alkylated protein returns
to the FTV, where it awaits mixing with an enzyme and incubation in the
30°C reaction coil. The digested protein is then ready for LC injection
and mass spectrometry detection.

\textbf{Synthetic Process Parameters Monitoring and Product Quality
Assessment by the A2P2 System}

For synthetic applications, the A2P2 system can carry out reaction
quenching, dilution, and chemical reactions before preparing the samples
for LC or LC-MS injection. An analyst can manually deliver samples to
the autosampler or use an automatic sampling system to do so. If
additional sample preparations are required, two reaction coils, each
with different temperature settings, are available for pre-injection
sample preparation. If desalting is necessary, a desalting column can
replace one of the coil positions to fulfill this task.

\textbf{CONCLUSIONS}

We have developed and deployed the LC-MS and LC integrated \selectlanguage{greek}µ\selectlanguage{english}SI process
analyzer systems for online MAM and glycan measurements in addition to
designing the turn-key A2P2 system to accelerate development of
attribute-centric biotherapeutic manufacturing processes. Key design
considerations included instrument footprint reduction, faster result
turnaround, software-driven features, capability consolidation, and
operational simplification. These systems are designed to be simple to
use and enable rapid execution of complex assays that can inform process
development. Additionally, they can be readily integrated into a
strategy thereby enabling real-time, attribute-focused process
development, monitoring, and control. Rapid availability of critical
quality attribute data provides insights that can guide purposeful
planning of subsequent experiments. Efficiency gains through the
integration of fluidic miniaturization, autonomous diagnostics, and
advanced analytics are substantial and lead to efficient development of
processes that consistently manufacture high quality products.

\textbf{ACKNOWLEDGEMENTS}

The authors extend their appreciation to Rohini Deshpande and Gang Xue
for their invaluable inspiration, strategic guidance, and managerial
support in the development of the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer and the A2P2
systems. We acknowledge Neil Soice and Nitin Rathore for their guidance
from the Drug Substance and Drug Product perspectives in our PAT
projects. Our gratitude goes to Alicia Zeng for her pioneering work at
AMA (Amgen Massachusetts), where she implemented the \selectlanguage{greek}µ\selectlanguage{english}SI process
analyzer for real-time MAM data collection from a production bioreactor.
We also express our gratitude to the ATO (Amgen Thousand Oaks) Rapid
Analytics team members Eleanor Le, Jennifer Zapparelli, Danielle
Borchard, and Miranda Hutt for their assistance during the A2P2 system's
comparability testing phase. Gary Li, Da Ren, and Jason Richardson are
acknowledged for their strategic and technical input during the early
phase of A2P2 development for MAM, alongside Tom Dillon for his
expertise in LC software operation, which improved our understanding of
the SST and IRC algorithms.

\textbf{REFERENCES}

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  Ruzicka, J., Marshall, G. D. (1990) Sequential injection: a new
  concept for chemical sensors, process analysis and laboratory
  assays,\emph{Analytica Chimica Acta} , 237, 329-343.
  https://doi.org/10.1016/S0003-2670(00)83937-9
\item
  Tharmalingam, T., Wu, C. H., Callahan, S., \& T Goudar, C. (2015). A
  framework for real-time glycosylation monitoring (RT-GM) in mammalian
  cell culture. \emph{Biotechnology and Bioengineering} , 112(6),
  1146--1154. https://doi.org/10.1002/bit.25520
\end{enumerate}

\textbf{TABLES}

Table 1: High level example of process steps for MAM sample preparation
in the \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Process Step & Action\tabularnewline
\midrule
\endhead
Sampling & Aspirate 2 mg protein sample from source\tabularnewline
Affinity Cleanup & Protein retained, purified, and eluted from the
affinity column and deposit into the holding coil downstream of the
affinity column.\tabularnewline
Denaturation & Deliver the purified protein from the holding coil into
the 30C incubator. Add denaturation solution. Incubate for 10
minutes.\tabularnewline
Reduction & Add reduction solution into the 30C incubator. Incubate for
30 minutes\tabularnewline
Alkylation & Add alkylation solution into the 30C incubator. Incubate
for 20 minutes.\tabularnewline
Quenching & Add quenching solution. Incubate for 5
minutes.\tabularnewline
Desalting & Transfer the entire amount of mixture from the 30C incubator
to the desalting column. Capture the desalted protein through the 3-way
valve to the 40C incubator.\tabularnewline
Enzymatic Digestion & Add enzyme solution into the 40C incubator.
Incubate for 10 minutes.\tabularnewline
LC Injection & Deliver the digested protein mixture to the LC sample
loop and initiate LC-MS method.\tabularnewline
\bottomrule
\end{longtable}

Table 2: \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer high-level process steps for glycan map
sample preparation\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Process Step} & \textbf{Action}\tabularnewline
\midrule
\endhead
Affinity Capture \& Cleanup & Protein sample is captured, and its matrix
components are removed.\tabularnewline
Release of Glycan & Enzyme PNGase F is pushed into the affinity column
where purified protein resides and glycans are released into
interstitial space of the column. Released free glycans are removed from
the affinity column.\tabularnewline
Glycan Labeling & The release free glycans and glycan labeling reagent
are mixed, degassed, and heated inside the reaction coil where the
labeling reaction occurs.\tabularnewline
Cooling and Enrichment of the labeled glycans & The reaction mixture is
delivered to the cooling coil and labeled glycans are captured and
enriched in the PGC column. Excess reagent is removed as the
flow-through.\tabularnewline
Prepare for LC Injection & Labeled glycans are eluted from the PGC
column. The organic content of the PGC eluted sample is adjusted in the
mixing chamber before loading to the LC sample loop and initiate
HILIC-LC method for glycan quantitation.\tabularnewline
\bottomrule
\end{longtable}



\textbf{FIGURES}

\textbf{Figure 1} : The \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system for MAM sample
preparation. (a) The fluidic diagram, (b) \emph{Left} : The \selectlanguage{greek}µ\selectlanguage{english}SI process
analyzer system connected to a LC-MS system on the manufacturing
floor.\emph{Right} : Close up view of the upper compartment inside the
functionalized \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system.

\textbf{Figure 2} : The \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system for glycosylation
profile assay sample preparation. (a) The fluidic diagram,
(b)\emph{Left} : The \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system connected to a Waters
Acquity UPLC system in development lab. \emph{Right} : Close up to the
upper compartment inside the functionalized \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer system.

\textbf{Figure 3} : The A2P2 system. (a) The fluidic diagram, (b) A
configured A2P2 system utilizes a commercially available LC system. All
components are integrated vertically for efficient use of laboratory
footage. (c) The sample preparation unit operation beneath the
autosampler in the A2P2 system. (RxN Coil: reaction coil)

\textbf{Figure 4} : Engineering design of the FTP and its holder.
(a)\emph{Left} : FTV isometric view, cross-section of vial with arrows
indicating liquid flow. \emph{Middle} : FTV holder. \emph{Right} : Full
assembly of vial holder and FTV. (b) The FTV is a key part for the
breadth of functionalities in the A2P2 system. The extended gutter and
added lip to help prevent surface tension. Extrusion at the end of the
gutter is conveniently placed directly over the waste port.

\textbf{Figure 5} : The logical process developed by utilizing the LC
chromatography data system software to enable autonomous operation in
the A2P2 system. The uses of SST and IRC workflows enable autonomous and
self-directed sample preparation and/or assay execution. (LLOQ: lower
limit of quantitation, ULOQ: upper limit of quantitation, SEC: size
exclusion, SST: system suitability testing, IRC: intelligent run
control)

\textbf{Figure 6} : (a) The TIC readout from the LC-MS connected \selectlanguage{greek}µ\selectlanguage{english}SI
process analyzer system demonstrated good robustness over the course of
6 days continuous reference standard processing. (b) The real-time
product quality output from a production bioreactor. The trending curves
for deamidation and clips generated from the real-time MAM results can
be used for process decisions, in-process control, and product release.

\textbf{Figure 7} : (a) Glycan chromatogram generated from a reference
standard processed by the integrated HILIC UPLC \selectlanguage{greek}µ\selectlanguage{english}SI process analyzer,
full and zoomed view. (b) The performance of the system was verified by
daily processing of the reference standard for 32 days. (c) Continuous
glycan monitoring of a production bioreactor by the system over the
duration of entire upstream bioproduction process.

\textbf{Figure 8} : Capabilities of the A2P2 system. (a) Aggregation
assessment for biological products, (b) Charge variant profile for
biological products, (c) Glycosylation profile for biological products,
(d) Multi-attribute method for biological products. Out-of-scope flow
paths were masked for simplicity.

\selectlanguage{english}
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