Biological impurities &

their analysis
Teresa Domagala

Impurities - sources, detection and measurement 1

 Protein therapeutics differ from small molecule drugs
Small molecule medicines
Size & Structure
The active ingredient has a
chemical structure that is small
and simple
Manufacturing
Chemical synthesis readily scalable and robust.
Terminal sterilisation of the product is possible
Product quality
Small molecules manufacturing is less complex and
simple tests and controls are required to
demonstrate identity, quality, potency and purity
Stability
Small molecules are generally stable in different
formulations/ solvents and over a wide temperature
range
Impurities - sources, detection and measurement
Biological medicines
Biologics need to be made in
living cells. They are 100s-
1000s times larger and far
more complex
Product is produced in a unique living cell line. The
host is considered a source of intrinsic infectious
agents. Aseptic technique is required throughout
the process
Biological medicine manufacturing is more
complex and sophisticated tests and controls are
required to demonstrate identity quality, potency
and purity. Characterisation of process and
product related impurities is required.
Biological medicines are highly sensitive to
environmental changes including formulations
and temperature
2
 Process & Product related impurities (I)

Note For Guidance On Specifications: Test Procedures And Acceptance Criteria For
Biotechnological/Biological Products (CPMP/ICH/365/96) states:

Process related impurities, i.e., cell substrates (e.g., host cell

proteins, host cell DNA), cell culture or downstream processing.
Product-related impurities (e.g., precursors, certain degradation
products) are molecular variants arising during manufacture and/or
storage, which do not have properties comparable to those of
the desired product with respect to activity, efficacy, and safety.
Further, the acceptance criteria for impurities should be based on
data obtained from lots used in preclinical and clinical studies and
manufacturing consistency lots. Individual and/or collective
acceptance criteria for impurities (product-related and process-
related) should be set, as appropriate.

Impurities - sources, detection and measurement 3

 Process & Product related impurities (II)
PROCESS IMPURITIES
Adventitious viruses: demonstration of freedom from the agent
Host cell protein: <100pg/ therapeutic dose
Host cell DNA: <10ng/ therapeutic dose
Leached protein A: <ppm/ therapeutic dose
Fermentation requirements i.e. antifoam, selection agents

PRODUCT RELATED IMPURITIES

Aggregates
Breakdown products
Product variants, i.e. aberrant glycosylation, oxidation, deamidation,
denaturation, loss of C-terminal Lys (Mabs) etc.

Impurities - sources, detection and measurement 4

Process Related Impurities

Adventitious agents
Host cell protein
Host cell DNA
Leached protein A

5
 Viral Clearance

Cell lines are checked for viral contamination

Demonstration of viral clearance required for mammalian cell expressed

product
Design the process upfront to theoretically address the potential risks
Two orthogonal robust steps (e.g., low pH, nano-filtration,
solvent/detergent treatment, heat) typically included in the purification
process

Perform small scale clearance study that mimics the purification process
Spike Drug Substance with a model virus to demonstrate viral removal
by several logs beyond the potential load

Impurities - sources, detection and measurement 6

 Host Cell Protein (HCP)

USP <1132> Residual Host Cell Protein Measurements in

Biopharmaceuticals is due for publication in USP 38 (2015)

HCPs can vary in pI (~3-11) , hydrophobicity and display a wide range of

molecular weights (<5kDa to >250kDa) depending on host cell and
manufacturing method

Increasing pI
Historically, 2D IEF/ SDS-PAGE has been
used as a qualitative measure of HCPS

Increasing MW
Current methodology is to use immunoassay
methods

CHO HCP 2D IEF/SDS PAGE

(Western Blot)
Impurities - sources, detection and measurement 7
 Host Cell Protein (HCP) cont.

Schematic: HCP ELISA

Polyclonal anti CHO HCP

HRP conjugate
Performance Characteristics
Sensitivity
product HCP
Precision [intra & inter assay]
Recovery/ Interference
Polyclonal -CHO HCP
Specificity/ Cross reactivity

Impurities - sources, detection and measurement 8

 Host Cell DNA (HC-DNA)

Methods for quantification of host cell

DNA include: Schematic: qPCR
Pico Green analysis
Free dye is essentially non-fluorescent but on
binding to dsDNA, the dye exhibits >1,000 fold
fluorescence & signal is proportional to [dsDNA].
Hybridization
Labelled probes are bound to denatured and
immobilised HC-DNA. Signal is detected by
autoradiography or phosphor/fluorescence imaging
systems
Threshold assay
HC-DNA is a sample is complexed with a biotinylated
DNA binding protein and enzyme conjugated anti
DNA antibody. Enzyme activity is proportional to
[DNA]
qPCR (emerging industry standard)

Impurities - sources, detection and measurement 9

 Leached protein A

Most antibody/ Fc fusion protein therapeutics utilise an immobilised

Protein A [i.e. Mab Select SuRE ] capture step in the purification BUT
Protein A itself is a bacterial cell wall protein
Protein A [Staphylococcal Protein A, SpA] can react with IgG in the body causing
anaphylactic reactions thus it is important that antibodies be free of SpA

Similar to the detection of HCP,

Leached Protein A is quantitated
by ELISA
- The leached protein A much first be
dissociated from the antibody

Impurities - sources, detection and measurement 10

Product Related
Impurities

11
 Product related impurities

FDA has no specific guidance on the level of product related impurities in

a biological drug product

ICH Q6B Specifications; Test procedures and acceptance criteria for

Biotechnology/ Biological products, recognizes the heterogeneity of
Biological products

Impurities - sources, detection and measurement 12

 Impurity Decision Tree & Specifications

Common process impurities

Identify the impurity
include HMW & LMW species,
charge variants, oxidised species

Chromatographic & gel

Develop a suitable assay to measure the impurity
electrophoresis methods in
particular are widely used

Modifications may increase

the immunogenicity profile
of the molecule, increase
clearance rates or decrease
functional activity
Understand the capability of the process to control the
Assess the safety/ efficacy risk of the impurity
Ion exchange or hydrophobic
interaction and more recently
mixed mode chromatography
impurity are widely used to remove
impurities during DS
manufacture

Set meaningful specifications from the understanding

of critical process parameters that produce a product
with appropriate impurity characteristics

Impurities - sources, detection and measurement 13

 Analytical methods & their uses (I)

Gel permeation (GP) / Size exclusion (SE) HPLC mAU

DAD1F, Sig=215,8Ref=360,100(CEP37248\CEP37248STABILITY2011-01-1113-57-24\1AB-0402.D)
fragment

8.192

Are 8.890

.7
8.3
- Quantitation of HMW and LMW

826
624

a: 2
a: 3
Are
80

60
Aggregate

68
40

05.2
A6re.993
a: 8

09
70.8
A1re0.286
a: 5
20

47
58.9
A5re.899
a: 1
0

Ion Exchange HPLC

5 6 7 8 9 10 min

- Quantitation of charge variants commonly associated with

deamidation/ isomerisation events in antibodies

Reversed Phase HPLC

- Quantitative assessment of heavy & light
chain modifications of antibodies.

Impurities - sources, detection and measurement 14

 Analytical methods & their uses (II)

Peptide mapping/ MS analysis

N and C terminal truncations
specific amino acid modifications i.e. oxidation, deamdiation
confirmation of disulphide bonds

Carbohydrate analysis
glycans are released from the protein enzymatically, separated from
the protein scaffold and analysed using MS and or LC based techniques

15
Impurities - sources, detection and measurement
 Analytical methods & their uses (III)
Gel electrophoresis
Recent advancements are seeing traditional polyacrylamide gel
electrophoresis [SDS PAGE, IEF PAGE] being replaced by capillary
electrophoresis methods [CE-SDS, ic-IEF]
- These imaged capillary methods provide superior detection (signal to
noise), reproducibility & robustness over traditional quantitativePAGE
methods

Comparison of data from SDS PAGE (inset) and CE-SDS Product related impurity assignment for the CE-SDS
Normal vs heat stressed IgG electropherogram

Impurities - sources, detection and measurement 16

 Conclusion

When dealing with biological therapeutics

The major components of the impurity profile can be either:
Process related impurities
Product related impurities

Acceptable limits have been established for (most) process related impurities
however the onus is on the manufacturer to set specifications around product
related impurities based on:
Pre-clinical and clinical safety studies
The capability of the process to remove the impurities
Related products/ historical data

The manufacturer must submit impurity specifications that reflect an

understanding of the phase of development , impurity risk and process
capacity to produce a product with a safe impurity profile

Impurities - sources, detection and measurement 17