View our webinar here: https://bit.ly/2lKNdWX Many different impurities are present in or generated during biotherapy manufacturing. This webinar will address how process contaminates can arise from raw input materials, occur as residual processing agents, or form as reaction by-products. We will review strategies within product characterization to de-risk the manufacturing process, including the use of routine and high complexity assays; and the recommended testing to meet regulatory requirements for clinical submission. Learn methods to avoid costly pitfalls and implement procedures to expedite product quality decisions at critical junctures in your development plan. We will discuss two types of therapies: Cell & Gene Therapies Polyethylenimine (PEI) is a transfection agent used in nearly all cell and gene therapy products. We will review the regulations and the liquid chromatography with charged aerosol detection (LC-CAD) methodology to demonstrate PEI removal during the production process. Monoclonal Antibodies (mAb) and Cell & Gene Therapies During mAb manufacturing and inherent to Cell & Gene Therapies, a significant proportion of process impurities arise from the host cell used to express the drug. Host cell protein (HCP) impurities, present at PPM-levels, are a major immunogenicity risk because they can elicit an unpredictable immune response in patients. We will review why their complex and diverse nature makes them challenging to monitor, and theho best practices, specifically HCP identification by mass spectrometry, for detection. Learning points: 1. Accurate detection and characterization of residual PEI in cell and gene therapy products 2. Effective detection and characterization of residual host cell proteins (HCP) in mAbs 3. Available technology and assays for quantifying process impurities 4. Current regulatory requirements for detecting, quantifying, and removing process impurities during biotherapy manufacturing

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Process Impurities:
Don’t Let PEI or HCP Derail
Your Biotherapy
Janice Lord & Omar Lamm
September 12, 2019

2. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Process Impurities:
Don’t Let PEI or HCP Derail
Your Biotherapy
Janice Lord & Omar Lamm
September 12, 2019

3. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada.

4. The manufacturing of a biotherapy involves multiple inputs,
ranging in complexity from a transfection reagent to the host
cell in which the therapy is manufactured. All of these process
components should be removed prior to the therapy being
available for human use.
During today’s webinar we will review strategies within product
characterization to de-risk the manufacturing process for cell
and gene therapies and monoclonal antibodies (mAbs).
We will focus on how we effectively detect and characterize:
• polyethylenimine (PEI), used in cell and gene therapy
products; and
• host cell proteins (HCPs), a factor in mAb therapies and
cell and gene therapies.
Introduction

5. Detection and
Quantitation of PEI
in cell and gene
therapy products
Janice Lord, Analytical Chemistry Lab Manager

6. Agenda
PEI Description and Use
Analytical Challenges
1
2
4
3
Testing guidelines / guidance
5
Our Method for PEI
detection & quantification:
UHPLC-CAD
GMP Processes / Method
Validation

7. 7
Cell and Gene Therapy: Safety & Characterization Testing
Plasmid or
Virus
Master Cell Bank (MCB)
Working Cell Bank
(WCB)
Process Development
(Growth/Production/Modification)
Plasmid Stock
Master/Working Virus Bank
(MVB/WVB)
Drug Substance
Drug Product
Cell
Identity
Safety
Stability
Lot Release
Testing
QA/QC
In-
Process
Testing
Identity
Purity
Safety
Identity
Purity
Safety

8. Gene transfection agent
 Spontaneously adheres to and condenses DNA to
form spherical complexes that are readily
endocyclosed by cells. Once inside the cells, DNA
is released into the cytoplasm
 Released DNA undergoes transcription and
translation giving rise to protein products or
 Is used as a template for production of viral
vectors
 Free PEI can cause cell death and induce cellular
stress responses
8
Description and Uses
PEI
Other uses
 Polymeric coating for nanoparticles to deliver
anti-cancer agents
 Increasing attachment of weakly anchoring cells
in cell culture
Cationic (positively charged) polymer with surfactant properties

9. 9
Testing Guidelines / Regulations for PEI
Regulatory requirements
ICH Q6B (1999): 2.3 Process controls / 2.3.1 Process-related considerations
• Evidence is required to show that PEI is removed (or controlled at an acceptable level) in
cell and gene therapy products.
When to test for PEI
• Development of manufacturing process
• Not needed once evidence demonstrating PEI removed/controlled
• As part of development, monitor PEI levels throughout the manufacturing process to
confirm removal of PEI.
• Justification for exclusion from LRT is provided if PEI removal is confirmed

10. Method development needs to overcome all these challenges
PEI does not contain
chromophores
PEI has multiple
positive charges
PEI binds
irreversibly to
silica based
columns
10
PEI is not an easy molecule to analyze
Analytical Challenges: Why you Should Outsource
Interference occurs
with
peptides/proteins
PEI is not a single
molecule and exists
in several forms
PEI has poor
aqueous
solubility and is
poorly soluble at
high pH

11. PEI
 What form and Molecular Weight is used
 Who is the supplier
 Obtain a sample of the PEI used from the supplier
 Solubility information
Sample Matrix
 What other components are in the sample matrix
 How much of each component is in the matrix
 If the method is to be used for IPC, consider matrix components
at each stage
 Obtain/make placebos
Outsourcing
 Avoid need for capital expenditure
 Choose partner with established latest technology and expertise
11
Analytical Challenges: Why you Should Outsource
Key information to know to get started measuring PEI
Repeating units
Linear
Branched

12. PEI Detection Types
12
PEI does not have chromophores i.e. does not absorb uv or visible light
Need alternative modes of detection
 Evaporative light scattering detection (ELSD)
 LC eluent is nebulized, mobile phase is evaporated from the droplets leaving particles. Particles
scatter light in an optical cell. The amount of light scattering is related to the mass of the particles
 May lack sensitivity when looking for residual levels of PEI
 Mass spectrometry
 Separates molecules according to their mass to charge ratio
 Major advantage is that as well as detection and quantification, MS can be used to confirm identity.
 Disadvantages: cost and time
 Complexation with UV absorbing moiety, then UV detection
 Reaction has to be specific for PEI
 Charged aerosol detection (CAD)
 Similar to ELSD except the dry particles are charged using a high voltage then collected and
measured with an electrometer.
 Provides near uniform response regardless of the analyte

13. Method: UHPLC-CAD
PEI Detection Workflow
13
PEI extracted from sample
matrix
Extract separated into
component parts via High Liquid
Chromatography (UHPLC)
The eluent from the UHPLC is
nebulized in the CAD and heated to
create dry particles.
AUC for PEI signal is used to
determine concentration
The particles are charged using
high voltage. The charged
particles are collected and
measured with an electrometer.

14. Separate or remove matrix components
Sample Preparation
14
Points to consider
Relative
Solubility of
components
Enzymatic
digestion
Solid phase
extraction
Precipitation
Centrifugation

15. 15
Summary
PEI Detection through UHPLC-CAD
Increasing Regulatory scrutiny
Sensitive, robust and validated methods are needed to determine residual levels of PEI in
CGT products
Outsourcing partner
Avoid the need for capital investment in new equipment and could be a quicker route to
reliable results
Other impurities that fit this method under
consideration
Triton, Tween, etc.

16. Host Cell Proteins:
Identification by
mass
spectrometry
Omar Lamm, Product Characterization Technical Support

17. We will now focus on how we effectively detect and characterize host
cell proteins (HCPs), a factor in mAb therapies and cell and gene
therapies.
Host Cell Proteins: Identification by Mass Spectrometry
HCP impurities, present at PPM-levels, are a major
immunogenicity risk because they can elicit an unpredictable
immune response in patients.
We will review why their complex and diverse nature makes them
challenging to monitor and the best practices, specifically HCP
identification by mass spectrometry, for detection.

18. Agenda
HCP – Potential impacts on biotherapies
Why traditional HCP identification approaches are not satisfactory
1
2
3
Progressive techniques for HCP
identification: HCP by MS

19. Immunogenicity
• The ability of a particular substance, such as an
antigen or epitope, to provoke an immune
response in the body of a human and other
animal.
Lipases
• Enzymatic cleavage of fats or fatty acids
(polysorbate)
Proteases
• Enzymatic cleavage of proteins
19
Host Cell Proteins
How Can They Impact my Biotherapy?

20. Immunogenicity—case presented at BPI 2016 forum featured a
product expressed by a customized proprietary CHO cell line that elicited
anti-HCP antibodies in patients during clinical trials.
• Known offenders
• Various in silico options hitting the market
20
Host Cell Proteins
Immunogenicity
• USP General Chapter <1106> for a more in-depth discussion of factors influencing
immunogenicity. When patients receive more than one biotherapeutic protein with their
associated impurities, immunogenicity is an even greater concern.

21. One such CHO protein is phospholipase B-
like 2 (PLBL2), which has been observed
at different levels in mAbs produced using
platform processes.
Levels >10 ng/mL caused nonlinear
dilutions in a platform HCP ELISA assay.
Recently, degradation of polysorbate
surfactant used in protein formulations
was found to result from the presence of
trace phospholipase levels in mAbs
produced by CHO cells and enzymes from
a human cell line.
21
Host Cell Proteins
Lipase Activity

22. Understanding the Concern
ICH and USP
22
International Council for Harmonization (ICH) of Technical Requirements
• Drug Substance Critical Quality Attributes
For biotechnological/biological products, impurities may be process-related or product-related. Process-
related impurities include: cell substrate-derived impurities (e.g., Host Cell Proteins (HCP) and
Host Cell DNA.
• Residual Host Cell Protein Measurement in Biopharmaceuticals
Targets, alert limits, and reject limits—requires biological products to be “...free of extraneous
material except that which is unavoidable...”
Safety considerations in setting limits—high-dose products may contain a higher mass of residual
HCPs per dose but the potential effect is not necessarily predictable of a clinical outcome. In some
cases, very low levels of certain HCPs have been shown to have clinical effects.
United States Pharmacopeia (USP)

23. Agenda
HCP – Potential impacts on biotherapies
Why traditional HCP identification approaches are not
satisfactory
1
2
3
Progressive techniques for HCP
identification: HCP by MS

24. Current Standard of HCP Measurement - ELISA
24
Sensitivity but lack of specificity
• Polyclonal anti-HCP antibodies recognize a very wide range of possible HCPs
• The first proteomic study of CHO-K1 identified about 6,000 proteins1.
• Typically, a single HCP numerical value is reported for such assay results, representing the ratio of HCPs
(ng) to product (mg). That value can reflect a single protein or a collection of several such impurities.
• Furthermore, if a potentially significant copurifying impurity is nonimmunogenic (or weakly
immunogenic), then the assay could miss it.
• Likewise, if a copurifying impurity is highly immunogenic, the assay could overquantify its value. For
these reasons, orthogonal analytical methods often are used to ensure product purity2.
1. Baycin-Hizal D, et al. Proteomic Analysis of Chinese Hamster Ovary Cells. J. Proteome Res. 11, 2012: 5265–5276.
2. Zhu-Shimoni J, et al. Host Cell Protein Testing By ELISAs and the Use of Orthogonal Methods. Biotechnol. Bioeng. 111(12) 2014: 2367–2379.

25. Current Standard - ELISA
25
Regardless of the immunization or purification strategy chosen, both regulators and industry consider
it critical to characterize the resulting anti-HCP reagent’s degree of immunospecificity.
Key characterization elements include:
• Assessing the reagent’s lack of cross-reactivity with the protein product itself
 Assessing its ability to detect low-abundance and/or low–molecular-weight HCP species
• Assessing the detection of HCP subpopulations that are enriched in downstream steps
• Demonstrating the percentage of the total host cell proteome that the mAbs can detect.

26. 26
• Clone selection
• Downstream Optimization
- Differential binding and elution
• Scale-up
- It wasn’t there before?
• Tech Transfer
- Minor differences could affect copurification
• Reagent control and bridging
• Dilutional nonlinearity
26
Non-Specific ELISA – What are we Missing?

27. Agenda
HCP Sources and Concerns
Why traditional HCP identification approaches are
not satisfactory
1
2
3
Progressive techniques for HCP
identification: HCP by MS

28. 28
HCP by Mass Spec – Comprehensive ID
The Goal –
100% ID of all HCPs above
a certain threshold
(e.g. 10ppm)
Challenges:
• Complex mixtures (harvested cell-culture)
• Process related impurities (e.g. Protein A)
• Sample prep related impurities (e.g. PNGase F,
skin keratins, etc.)
• PTM variations as well as incomplete cleavages
• Database
• API signal intensity

29. Approach:
• Database – host cell proteome, etc.
• Sample prep
• LC/MS (DDA)
• Data analysis
• Assignments
• Focus on ID rather than absolute quantitation
(semi-quantitative)
• Minimize or eliminate unassigned masses
29
HCP by Mass Spec – Comprehensive ID

30. • 1D LC-MS assay for individual HCP identification, quantification, and monitoring
- Sample preparation – denaturation, reduction and alkylation, digestion
• First analysis step is the HCP discovery assay via data independent MSE acquisition
using 90 minute peptide separations
• Second analysis step samples are analyzed by higher-throughput HCP monitoring
assays using MSE acquisitions with 30 minute peptide separations
• Data acquired in RAW format and processed e.g. with Progenesis QI for proteomics
• In summary – The Discovery Assay populates the database and the Monitoring
Assay references this database for subsequent purification steps
HCP Detection Workflow
30

31. Utility of multiple fractionations for the identification of
low-abundance peptides from a complex mixture
31
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338939/

32. Determining which HCPs qualify for removal
32
HCPs identified across
six PTG1 preparations.

33. Roundtable Discussion – CASSS 2018
33
Quantitation/Detection:
• Some companies are removing the mAb to gain more sensitivity of
HCPs.
• A lot of people are using MRM for quantitation of HCPs and Orbitraps
and/or QTOFs for identification.
• For relative quantitation for HCPs, some people use abundance
obtained during HCP identification analysis and for absolute HCP
quantitation, they use a quantitation through a targeted MRM.

34. HCP by Mass Spec – Quantitation by MS
34
Do we need absolute quantitation on all HCPs?
• No – let’s focus on the problematic ones
• Minimal development time
• SLI with MRM for wide dynamic range and low RSD – choose signature peptides
• Typical to see multiple per assay
• Waters Corporation published absolute quantitation for PLBL2 in mAb at ppb level
using Xevo TQ-S Tandem Quadruple MS

35. Identify HCP(s) of interestDependent on various reagents
Assay development (> 6 months)Assay development ($100k+)
What’s Next? HCP – Protein Specific Quantitation by ELISA
1
2
4
3
35

36. Roundtable Discussion – CASSS 2018
36
What is the future?
Most agree that we need both (LC-MS/MS and ELISA) because they are very
powerful together. How do we prove the robustness of LC-MS/MS to the FDA if
all industry is using different instruments, samples preparation, and software?
It may be difficult to align HCP analysis by LC-MS/MS because there are so
many different modalities: Gene therapy, monoclonal antibodies, bi-specifics,
vaccines, and fusion proteins. Everybody agrees that there’s a prompt need for
FDA to provide guidance for HCP analysis.

37. 1. Risk mitigation
 Scale up
 Tech transfer
2. Trend of expectation
 Submission trends
 USP
 Response letters
3. Process Characterization
37
HCP by Mass Spec
Why Should we Adopt

38. Cell and Gene Therapy
• Accurate detection and characterization of residual PEI in cell and gene therapy
products
Monoclonal Antibodies
• Effective detection and characterization of residual host cell proteins (HCPs)
Combined Learnings
• Available technology and assays for quantifying process and product impurities
• Current regulatory requirements for detecting, quantifying, and removing
impurities during biotherapy manufacturing
Today’s Take-Aways for Product Characterization
38

39. Gillian.Barr@milliporesigma.com
Janice.Lord@milliporesigma.com
Gillian Barr
Janice Lord
Analytical Chemistry Lab Manager
Omar Lamm
Product Characterization Technical Support
Omar.Lamm@milliporesigma.com
Thank you!