Immunoassays for Process-related Impurities
                     Immunoassays for 
 Process-related 
Impurities
Immunoassays for the detection of
process-related impurities in biologics
Creative Diagnostics provides highly sensitive and specific ELISA Kits for monitoring
process-related impurities in biologics
Content
Background ............................................................................................................................................................................................3
Cell substrate-derived impurities ...............................................................................................................................................4
Host cellular DNA..........................................................................................................................................................4
Ralated Product....................................................................................................................................................................4
Host cell proteins ..........................................................................................................................................................5
Ralated Product....................................................................................................................................................................6
Cell culture-derived impurities ....................................................................................................................................................6
Culture Media Components........................................................................................................................................ 7
Ralated Product.................................................................................................................................................................... 7
Antibiotics..................................................................................................................................................................... 10
Ralated Product.................................................................................................................................................................. 10
PEG .................................................................................................................................................................................. 11
Ralated Product.................................................................................................................................................................. 12
Downstream-derived impurities ................................................................................................................................................ 12
Ralated Product.................................................................................................................................................................. 12
Features.................................................................................................................................................................................................. 14
Contact ................................................................................................................................................................................................... 14
Immunoassays for process-related
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Background
Biopharmaceuticals are a class of drugs generally produced in living organisms that can be used for the 
 treatment of a wide variety of diseases. Biopharmaceutical samples include hormones, enzymes,  
monoclonal antibodies, vaccines, and blood factors, and each presents different challenges during drug  
development and manufacturing, one of which is the presence of process-related residual impurities 
and  their levels in the final biopharmaceutical drug product.
Fig. 1 The classification of biopharmaceuticals into three main categories, namely, amino acids, 
nucleic  acids, and vaccines. (Rasmussen, A. S. B., et al. 2021)
Process-related impurities are basically any chemicals or biological entities expected to be present in a 
 manufacturing process that is either not completely removed during purification or not desired to be 
in  the API (Active pharmaceutical ingredient) or the final drug product. Process-related impurities can 
be  divided into three categories:
 Cell substrate-derived impurities (e.g., proteins/nucleic acids derived from the host organism, such as 
 host cellular DNA).
 Cell culture-derived impurities (e.g., inducers, antibiotics, serum and other media components).
 Downstream-derived impurities (e.g., enzymes, chemical and biochemical processing reagents—  
cyanogen bromide, reducing agents, inorganic salts, solvents etc.).
Fig. 2 The biopharmaceutical manufacturing technology flowchart exemplifying the upstream 
and  downstream bioprocess. (Jozala, A. F., et al. 2016)
Immunoassays for process-related
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Effective removal of process-related impurities or process residues is very important for pharmaceutical  
and biopharmaceutical development. To meet the requirements for monitoring and controlling process-  
related impurities and contaminants in the biological manufacturing process as described in the ICH 
Q6B  Biomolecule Production Guidelines, Creative Diagnostics has developed a series of highly sensitive 
ELISA  kits to analyze impurity residues. These kits are suitable as good impurity detection tools used in  
biomanufacturing processes.
Cell substrate-derived impurities
During the manufacturing of biopharmaceuticals, some non-product, host cell-derived materials are  
inevitably introduced into the process stream. The major process-related safety concerns are residual 
 host cellular DNA and residual host cell proteins (HCPs), which must be removed by purification  
processes.
Host cellular DNA
Since host cellular DNA may contain oncogenic or tumorigenic sequences, both in terms of quantity 
and  size, in the genetically engineered viruses, it is important to ensure that such process-related 
impurities  are kept to a minimum. While several methods are available to measure very low levels of 
residual host  cellular DNA (e.g, DNA probe hybridization, Threshold® single stranded DNA binding 
analysis),  quantitative polymerase chain reaction (qPCR), also known as real-time PCR, has become the 
most  widely used method.
Fig. Flowchart of the direct qPCR approach. (Peper, G., et al. 
2014)
Ralated Product
Cat. No Product Name / Service Sensitivity
DEIA-BY050 CHO DNA residue ELISA Kit 3 ppt
DEIA-BY051 EN.AcorelisDidue ELISA Kit 0.03 ppb
DEIA-BY052 Human DNA residue ELISA Kit 0.03 ppb
DEIA-BY053 Human residual DNA fragment Assay Kit 0.03 ppb
ppb-level quantitative detection -- Fluorescence staining /  
HCD-S001 -
Threshold Assay / qPCR Assay
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Host cell proteins
The primary safety concern with HCPs in biopharmaceuticals is their potential to induce anti-HCP  
antibodies that could lead to clinical effects in patients. In addition, HCPs may act as adjuvants and  
induce anti-drug antibodies that may affect the safety or efficacy of the drug. HCPs can also have a  
direct effect on the quality of the product itself. For example, proteolytic HCPs, even in minute 
amounts,  can cleave the desired recombinant protein or monoclonal antibody over time, reducing or 
eliminating  biological potency or altering stability. While several methods are available to measure 
trace amounts of  residual HCPs (e.g., two-dimensional HPLC, mass spectrometry), HCP immunoassays 
(typically the  enzyme-linked immunosorbent assay, ELISA) remain the most widely used method.
Fig. A typical sandwich ELISA for host cell proteins
Immunoassays for process-related
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Ralated Product
Host Cat. No Product Name
DEIABL475 A549 HCP ELISA Kit
DEIABL476 BHK HCP ELISA Kit
DEIABL478 CHO Host Cell Protein ELISA 
Kit  DEIABL482 HEK 293 HCP ELISA Kit  
DEIABL484 HeLa HCP ELISA Kit
DEIABL486 MDCK HCP ELISA Kit
Mammalian DEIABL487 MRC5 HCP ELISA Kit
DEIABL488 NS/0 HCP ELISA Kit  
DEIABL490 PER.C6 HCP ELISA 
Kit
DEIABL496 SP2/0 Host Cell Protein ELISA 
Kit  DEIABL498 Vero Cell Host Cell Proteins
DEIASL149 HEK 293T HCP ELISA Kit  
DDEEIIAAB-BLY409112 PCicAhiPa  Cpealsl tLoirnise  HHCCPP  EELLIISSAA  Kit
Yeast
DEIABL494 Ser. ecvisiae HCP ELISA Kit
Kit
DEIABL481 CE.PcEoLliIH Kit
SA
DEIABL485 L. lactis HCP ELISA Kit
Bacterial
DEIABL489 P. fluorescens HCP ELISA 
Kit  DEIABL493 S. aureus HCP ELISA Kit
Insect DEIABL495 SF9 HCP ELISA Kit
Cell culture-derived impurities
The cell culture medium provides the complex mixture necessary for cell survival and biopharmaceutical 
 production. Different culture media are composed of amino acids, vitamins, sugars, and inorganic salts.  
However, cell culture media may also include serum (e.g., fetal bovine serum), growth factors (e.g.,  
insulin- like growth factor), selective agents (e.g., methotrexate), antibiotics, and process-enhancing
Immunoassays for process-related
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agents.
Culture Media Components
Serum typically contains albumin, immunoglobulins, and a rich mixture of other proteins. When these  
components are present as residual impurities, immunogenicity should also be considered. For example,  
for cell culture produced vaccines, extraneous proteins known to be capable of causing allergenic 
effects  in humans should not be added to a final virus medium of cell culture-derived vaccines intended 
for  injection.
Insulin and insulin-like growth factor-1 (IGF-1) are widely used growth factors to delay apoptosis in  
mammalian cell culture. However, growth factor residuals in biopharmaceuticals may become a 
concern  for affecting the drug activity.
Ralated Product
Analyte Cat. No Product Name Sensitivity
Insulin DEIA-BY021 Insulin ELISA Kit 0.125 ppb
DEIA9947 Human ALB(Albumin) ELISA Kit 0.5 ppb
DEIA605 Bovine ALB ELISA Kit 0.69 ppb
Albumin DEIASL274 Rabbit anti-BSA IgG ELISA Kit 1:200
DEIASL275 Human anti-BSA Antibody ELISA Kit 1:200
DEIA1523 Transferrin Human ELISA Kit 1.5 ppb
Transferrin
DEIA7585 Bovine Transferrin ELISA Kit 0.2 ppb
DEIA611 Bovine IgA ELISA Kit 1.37 ppb
DEIA612 Bovine IgG ELISA Kit 3.439 ppb
DEIA613 Bovine IgG1 ELISA Kit 1 ppb
DEIA614 Bovine IgG2 ELISA Kit 1 ppb
DEIA615 Bovine IgM ELISA Kit 1.37 ppb
Species-Specific
Immunoglobulin
DEIASL056 Camel IgG ELISA Kit 1.17 ppb
DEIA-BY033 Cat IgG ELISA Kit 0.8 ppb
DEIA-BY034 Cat IgM ELISA Kit 3.1 ppb
DEIA627 Chicken IgA ELISA Kit 15.63 ppb
DEIA3658 Chicken IgG (IgY) ELISA Kit 1.56 ppb
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DEIA629 Chicken IgM ELISA Kit 1.56 ppb
DEIA-BY035 Cow IgA ELISA Kit 3.91 ppb
DEIA-BY036 Cow IgG ELISA Kit 0.78 ppb
DEIA-BY037 Cow IgM ELISA Kit 15.63 ppb
DEIA7614 Canine IgA ELISA Kit 0.02 ppb
DEIA7616 Canine IgG ELISA Kit 0.5 ppb
DEIA7617 Canine IgM ELISA Kit 0.2 ppb
DEIA631 Dog IgA ELISA Kit 3.91 ppb
DEIA632 Dog IgE ELISA Set 0.78 ppb
DEIA633 Dog IgG ELISA Kit 2.5 ppb
DEIA688 Human IgA ELISA Kit 1.875 ppb
DEIA9467 Human IgE ELISA Kit 6.25 ppb
DEIA1480U Human IgG ELISA Kit 0.78 ppb
DEIA5459 Human IgG1 ELISA Kit 0.94 ppb
DEIA691 Human IgM ELISA Kit 0.94 ppb
DEIA7713 Horse IgA ELISA Kit 7.81 ppb
DEIA7715 Horse IgG ELISA Kit 3.13 ppb
DEIA647 Horse IgM ELISA Set 15.63 ppb
DEIA640 Goat IgG ELISA Kit 7.81 ppb
DEIA-BY038 Goat IgM ELISA Kit 12.5ppb
DEIA7756 Guinea pig IgG ELISA Kit 3.13 ppb
DEIA-BY039 Guinea Pig IgM ELISA Kit 3.91 ppb
DEIABL406 Monkey IgA ELISA Kit 3.91 ppb
DEIABL407 Monkey IgG ELISA Kit 3.91 ppb
DEIABL408 Monkey IgM ELISA Kit 3.91 ppb
DEIA-BY041 Monkey IgE ELISA Kit 5 ppb
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DEIA-BY042 Monkey IgG1 ELISA Kit 6.25 ppb
DEIA651 Mouse IgA ELISA Kit 0.93 ppb
DEIA652 Mouse IgE ELISA Kit 1.56 ppb
DEIA8704 Mouse IgG ELISA Kit 7.81 ppb
DEIA6402 Mouse IgG1 ELISA Kit 1.56 ppb
DEIA655 Mouse IgG2a ELISA Kit 0.195 ppb
DEIABL411 Mouse IgG2b ELISA Kit 0.195 ppb
DEIA658 Mouse IgG3 ELISA Kit 0.78 ppb
DEIA6405 Mouse IgM ELISA Kit 7.81 ppb
DEIA663 Pig IgA ELISA Kit 4.69 ppb
DEIA664 Pig IgG2b ELISA Kit 1.37 ppb
DEIA665 Pig IgM ELISA Kit 15.6 ppb
DEIA-BY054 Pig IgG ELISA Kit 1.56 ppb
DEIA-T6101 Porcine IgG ELISA Kit 0.5 ppm
DEIA8497 Rabbit IgA ELISA Kit 0.281 ppb
DEIA8498 Rabbit IgG ELISA Kit 3.13 ppb
DEIA8499 Rabbit IgM ELISA Kit 7.81 ppb
DEIA674 Rat IgA ELISA Kit 2.34 ppb
DEIA675 Rat IgE ELISA Kit 1.56 ppb
DEIA676 Rat IgG ELISA Kit 1.95 ppb
DEIA681 Rat IgM ELISA Kit 1.95 ppb
DEIA-BY043 Rat IgG1 ELISA Kit 15.63 ppb
DEIA-BY044 Rat IgG2a ELISA Kit 7.81 ppb
DEIA-BY045 Rat IgG2b ELISA Kit 1.58 ppb
DEIABL420 Sheep IgG ELISA Kit 15.63 ppb
DEIA-BY046 Sheep IgA ELISA Kit 0.156 ppm
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DEIA-BY047 Sheep IgM ELISA Kit 31.25 ppb
DEIA-BY048 Turkey IgG ELISA Kit 1.563 ppb
DEIA-BY049 Turkey IgM ELISA Kit 3.13 ppb
Antibiotics
In the cell culture process, the use of appropriate concentrations of antibiotics can prevent 
contamination  and the morphological or physiological changes caused by contamination. In addition, 
certain antibiotics  are also used to screen transfected/genetically modified cells. Therefore, antibiotics 
are common cell  culture-derived impurities in the biomanufacturing process. The following products can 
be used to detect  antibiotic impurities in cell culture medium, fermentation fluid, vaccine supernatant, 
DNA TE buffer and  other samples with high sensitivity, and are suitable for high-throughput quantitative 
screening of  samples.
Ralated Product
Analytes Cat. No Product Name Sensitivity
Carbenicillin DEIA-BY013 Carbenicillin ELISA Kit Under development
Chloramphenicol DEIA018 Chloramphenicol, CAP ELISA Kit 0.025 ppb
Colistin DEIA042 Colistin ELISA Kit 1.5 ppb
Geneticin DEIA-BY014 Genetici ELISA Kit Under development
Gentamicin DEIA047 Gentamicin ELISA Kit 0.1 ppb
DEIA048 Kanamycin ELISA Kit 0.5 ppb
High Sensitivity Kanamycin ELISA Test

Kanamycin DEIA-WZ048V 0.2 ppbKit
DEIA048V Kanamycin ELISA Kit 0.05 ppb
Meropenem DEIA-BY015 Meropenem ELISA Kit Under development
Methotrexate DEIA-XYZ209 Methotrexate ELISA Kit 0.16 ppb
DEIABL-QB24 Penicillin ELISA Kit 0.1 ppb
Penicillin
DEIABL-QB25 Benzyl penicillin ELISA Kit 0.1 ppb
Puromycin DEIA-BY016 Puromycin ELISA Kit Under development
Streptomycin DEIA020 Streptomycin ELISA Kit 0.1 ppb
Tetracycline DEIA046 Tetracyclines ELISA Kit 0.2 ppb
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Thiaphenicol DEIA-BY017 Thiaphenicol ELISA Kit Under development
Tobramycin DEIA-BY018 Tobramycin ELISA Kit Under development
Vancomycin DEIANJ11 Human Vancomycin ELISA Kit 0.5 ppb
(Catalog number marked with   are hot products)
After many tests, we obtained the parameters of the kit, as shown in the following Data 
Example:
Cat. No Product Name Detection Range IC50 CV Recovery Linearity
DEIA048 Kanamycin ELISA Kit 0.5-40.5 ppb 1.2 ppb ≤10% 81.2% to 102% 0.99
Cat. No Product Name Detection Range IC50 CV Recovery Linearity
DEIA047 Gentamicin ELISA Kit 0.1-8.1 ng/mL 0.442 ppb ≤10% 86% to 119.7% 0.99
PEG
The modification of biopharmaceutical molecules by covalent conjugation of polyethylene glycol (PEG)  
molecules is known to improve the pharmacological and pharmaceutical properties of proteins and other 
 large molecules. Frequently, one end of the polymer is capped with a methoxy group (mPEG) to 
prevent  unwanted cross-linking during conjugation. Residual impurities associated with such processing 
additives  must also be removed from the API or the final drug product.
Immunoassays for process-related
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Ralated Product
Category Cat. No Product Name Sensitivity
High Sensitivity Polyethylene Glycol 
DEIA615 0.064 ppb
For detection of 8  (PEG) ELISA Kit
PEGylated proteins
DEIABL235 mPEG ELISA Kit 0.3125 ppb
DEIA6159 Mouse anti-PEG IgG ELISA Kit 3.13 U/mL
DEIA6160 Mouse anti-PEG IgM ELISA Kit 6.25 U/mL
DEIASL085 Rat anti-PEG IgG ELISA Kit 6.25 U/mL
DEIASL086 Rat anti-PEG IgM ELISA Kit 3.13 U/mL
For detection of PEG DEIASL087 Monkey anti-PEG IgG ELISA Kit 1.56 U/mL
 antibody
DEIASL088 Monkey anti-PEG IgM ELISA Kit 6.25 U/mL
DEIA-BY029 Rabbit Anti-PEG IgG ELISA Kit 0.78 U/mL
DEIA-BY030 Rabbit Anti-PEG IgM ELISA Kit 1.56 U/mL
DEIASL243 Human Anti-PEG IgG ELISA Kit 0.78 U/mL
DEIASL244 Human Anti-PEG IgM ELISA Kit 3.13 U/mL
Downstream-derived impurities
Any API purification process can also introduce additional process-related impurities into the  
biopharmaceutical product. Potential downstream impurities such as enzymes, processing reagents (e.g., 
 cyanogen bromide, guanidine, oxidizing and reducing agents), leached metallic elements (e.g., leached  
from a metal-complex chromatographic resin), leached ligands (e.g., leached from a Protein A affinity  
column resin), and volatile organic solvents (from chromatography solvents), must all be evaluated.
Ralated Product
Protein A
Cat. No Product Name Sensitivity
DEIA6474 Protein A ELISA Kit 0.05 ppb
DEIA-BY019 Alkali-Resistant Protein A ELISA Kit 6.6 ppt
DEIA-BY020 Recombinant Protein A ELISA Kit 7 ppt
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Bioprocessing enzymes
Analyte Cat. No Product Name Sensitivity
Benzonase DEIA-JY2103 Benzonase ELISA Kit 0.05 ppb
Endonuclease DEIA-BY024 Endonuclease ELISA Kit 0.06 ppb
Vaccinia Capping
DEIA-BY031 Vaccinia Capping Enzyme ELISA Kit 0.05 ppb
Enzyme
2’-O- Methyltransferase DEIA-BY032 2’-O- Methyltransferase ELISA Kit 0.05 ppb
DNASE1 DEIANS026 DNase I ELISA Kit 0.05 ppb
T7 RNA Polymerase DEIANS032 T7 RNA Polymerase ELISA Kit 0.1 ppb
RNase Inhibitor DEIANS033 RNase Inhibitor ELISA Kit 5 ppb
Immunotoxicity
Analyte Cat. No Product Name Sensitivity
DEIA3834 KLH IgG (Mouse) ELISA Kit 6.25 U/mL
DEIASL066 KLH IgM (Mouse) ELISA Kit 6.25 U/mL
DEIASL067 KLH IgG (Rat) ELISA Kit 15.63 ppb
DEIASL068 KLH IgM (Rat) ELISA Kit 7.81 ppb
KLH
DEIASL069 KLH IgG1 (Monkey) ELISA Kit 0.94 ppb
DEIASL070 KLH IgM (Monkey) ELISA Kit 12.5 ppb
DEIA3833 KLH IgG (Guinea Pig) ELISA Kit 6.25 U/mL
DEIA-BY027 Human Anti-KLH IgG ELISA Kit 0.94 ppb
DEIA-BY028 Human Anti-KLH IgM ELISA Kit 31.25 ppb
DEIASL075 Rat Tetanus Toxoid IgG ELISA Kit 0.39 U/mL
DEIASL076 Rat Tetanus Toxoid IgM ELISA Kit 3.13 U/mL
Tetanus Toxoid DEIASL077 Monkey Tetanus Toxoid IgG1 ELISA Kit 0.078 ppm
DEIASL078 Monkey Tetanus Toxoid IgM ELISA Kit 3.91 ppb
Under
DEIASL264 Mouse Anti-Tetanus Toxoid IgG ELISA Kit
development
Immunoassays for process-related
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Under
DEIASL273 Rabbit Anti-Tetanus Toxoid IgG ELISA Kit
development
DEIA378 Human Tetanus Toxoid IgG ELISA Kit 0.001 IU/mL
DEIA2568 Human Anti-Tetanus Toxoid ELISA Kit 0.001 IU/mL
DEIASL081 Dinitropheno (Mouse) IgG ELISA Kit 3.125 U/mL
DEIASL082 Dinitropheno (Mouse) IgM ELISA Kit 3.125 
Dinitrophenol U/mL
DEIASL083 Dinitropheno (Rat) IgG ELISA Kit 3.125 U/mL
DEIASL084 Dinitropheno (Rat) IgM ELISA Kit 3.125 
U/mL
DEIASL079 Trinitrophenol (Mouse) IgG ELISA Kit 3.125 U/mL
Trinitrophenol
DEIASL080 Trinitrophenol (Mouse) IgM ELISA Kit 3.125 U/mL
Features
 High sensitivity—IC50 of the kit could reach ppb level.
 High specificity—Cross-reactivity with other antibiotics is less than 0.1%.
 High reproducibility—Coefficients of variation between and within batches were