ProteinAnalysisBlog https://www.alphalyse.com/proteinanalysisblog/ All about protein analysis Wed, 21 Dec 2022 12:01:49 +0000 en-US hourly 1 https://wordpress.org/?v=6.0.3 Quantifying HCP impurities in uncommon expression systems https://www.alphalyse.com/proteinanalysisblog/2021/10/29/uncommon-expression-systems/ https://www.alphalyse.com/proteinanalysisblog/2021/10/29/uncommon-expression-systems/#respond Fri, 29 Oct 2021 08:00:58 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2975 Question: We use a plant-based expression system to produce our immunotherapy-based biologics. Unfortunately, since the expression system is quite unusual, commercial ELISAs are unavailable. We want to analyze the host cell proteins (HCPs) in our drug substance (DS). Can we perform HCP analysis without developing a process-specific enzyme-linked immunosorbent assay (ELISA)? Answer: The short answer […]

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Question:

We use a plant-based expression system to produce our immunotherapy-based biologics. Unfortunately, since the expression system is quite unusual, commercial ELISAs are unavailable.

We want to analyze the host cell proteins (HCPs) in our drug substance (DS). Can we perform HCP analysis without developing a process-specific enzyme-linked immunosorbent assay (ELISA)?

Answer:

The short answer is yes; measuring and even quantifying Host Cell Proteins from unique expression systems is possible without developing a process-specific ELISA. One solution is to use liquid chromatography mass spectrometry (LC-MS).

Now, let’s go into more details.

The most common expression systems used to produce biologics are bacterial (e.g., E. coli) and mammalian (e.g., CHO and HEK) cell-based. However, other systems include plant-based, yeast, insect, viral, and cell-free systems.

Expanding host cell expression systems beyond E. coli, HEK, and CHO creates an increasing demand for new types of HCP-ELISAs. However, since these are not yet commercially available, many companies are forced to develop platform- or process-specific ELISAs. We will get back to the pros and cons of this in a minute.

Fortunately, there are less time-consuming options available such as LC-MS.

Essential reasons for analyzing HCPs in biologics

But why measure Host Cell Proteins (HCPs) at all?

HCPs constitute a significant class of process-related impurities and are particularly concerning in the drug manufacturing. This impurity poses a risk factor for reduced drug efficacy, toxicity, and long-term immunogenicity in drug recipients. Therefore, it is essential to demonstrate process consistency and purity of the final DS by documenting and monitoring any HCPs during process development and manufacturing [1].

In addition, identifying unwanted impurities guides manufacturers in adjusting purification steps and eliminating co-purifying impurities [1-3].

It is also a requirement from regulatory authorities.

Traditional methods for analyzing HCPs in biologics

The gold-standard method for HCP impurity analysis in biologics is ELISA. The advantages of ELISA include high sensitivity towards specific HCPs, simple and easy assay handling, short analysis time, and high sample capacity since you can analyze many samples simultaneously. Generic ELISAs are thus an adequate, semi-quantitative method to approximate the impurity level in the early processes [1, 4].

However, generic anti-HCP ELISAs can be scarcely available when using unique expression systems originating from, e.g., plants or insects. Such systems typically require the development of a platform assay to suit the specific cell line. Alternatively, a process-specific ELISA covers all potential impurities in the biologic manufacturing process and final DS.

Developing a platform assay includes many optimization steps such as antibody production and characterization and ELISA development and validation. Unfortunately, several issues can arise during these steps: Low antibody sensitivity or affinity, cross-reactivity or unspecific binding, high background signals, etc.

Typically you also need to produce a null-cell line for a mock fermentation – again, time-consuming and expensive.

A significant disadvantage of the platform and process-specific ELISA development is that it is time-consuming and may typically take at least a year. Also, it is not a one-time process: Once you use up the polyclonal antibody batches, new batches must be tested and validated. If these are unsuccessful, one must start with antibody production again [1-4].

Alternative to the development of a process-specific ELISA

As mentioned, other approaches are available to analyze HCPs in your biologics. The LC-MS method works independently of antibodies. It is therefore not as vulnerable to purification or manufacturing process changes that may require new antibody production.

The drug sample is prepared for analysis by digesting HCPs and drug substance proteins for LC-MS HCP impurity analysis. Then follows the first sample preparation steps, with adding intact protein standards. These standards act as quality controls and measures for the absolute quantification of unknown HCPs. After sample digestion, an ultra-sensitive LC-MS instrument analyzes the peptides, and a scientist searches the resulting data against databases of sequenced proteins to identify individual HCPs [3].

An advantage of the LC-MS approach is that it does not only provide the overall HCP impurity levels. The method also quantifies individual HCP impurity levels and gives detailed information about specific HCPs. E.g., name, accession number, pI, and molecular weight. The data makes it simpler for manufacturers to optimize purification steps and improve the purity of the DS. LC-MS can act as a quality tool to monitor batch-to-batch differences and control drug purity and efficacy [3].

To sum up, the LC-MS HCP analysis gives a more detailed quantitative and qualitative result than any ELISA. Since LC-MS results usually are obtained within a few weeks, such an assay is notably less time-consuming than developing a platform or process-specific ELISA.

Where do I look for more information?

If you are interested in learning more about how to monitor HCPs throughout purification steps without developing a process-specific ELISA, these webinars are an excellent place to start:

References

  1. U.S. Pharmacopeia National Formulary, 2016; May 1, USP 39 Published General Chapter <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals. https://www.usp.org/sites/default/files/usp/document/our-work/biologics/USPNF810G-GC-1132-2017-01.pdf
  2. Bracewell D. et al., Biotechnol Bioeng 2015 Sep;112(9):1727-37, DOI: 10.1002/bit.25628. The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control.
  3. Pilely K. et al., Anal. And Bioanal. Chem. 2021 Oct 1;1-12, DOI: 10.1007/s00216-021-03648-2. Monitoring process-related impurities in biologics-host cell protein analysis.
  4. Zhu-Shimoni J et al., Biotechnol Bioeng 2014 Dec;111(12):2367-79. DOI: 10.1002/bit.25327. Host cell protein testing by ELISAs and the use of orthogonal methods.

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How to select an HCP ELISA with sufficient coverage for regulatory documentation https://www.alphalyse.com/proteinanalysisblog/2021/08/25/selecting-best-elisa-kit/ https://www.alphalyse.com/proteinanalysisblog/2021/08/25/selecting-best-elisa-kit/#respond Wed, 25 Aug 2021 08:38:19 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2897 Question: How do I choose the best ELISA kit for my biologic? In other words, I would like to select the impurity assay with the highest coverage of the process HCPs, not only for harvest samples but also for the HCP impurities in the purified drug substance. Answer: Host Cell Protein (HCP) analysis is essential […]

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Question:

How do I choose the best ELISA kit for my biologic? In other words, I would like to select the impurity assay with the highest coverage of the process HCPs, not only for harvest samples but also for the HCP impurities in the purified drug substance.

Answer:

Host Cell Protein (HCP) analysis is essential in development and regulatory documentation of biologics.

Since specific HCPs are problematic, either as a potential risk factor for immunogenicity in patients or by affecting drug stability, biologics should be as free of residual HCPs as possible [1, 2].

Historically, most companies performed HCP analysis using anti-HCP ELISAs raised by immunizing animals with a protein lysate from the host cell line [1, 2].

Today, most companies also use orthogonal methods for HCP characterization, including liquid chromatography mass spectrometry (LC-MS), to quantify individual and potential problematic HCPs. Orthogonal methods are especially beneficial when there is no commercial kit on the market, or it is impossible to develop a good HCP ELISA.

Still, HCP ELISAs are valuable during process development, characterization, and validation [2].

(Learn more in this blog post).

Selecting the best ELISA kit: HCP antibody coverage analysis

Before a commercial ELISA kit or a custom-developed ELISA is considered sufficient and fit-for-purpose, you must perform an HCP antibody coverage analysis. This analysis determines how completely a population of polyclonal antibodies recognizes the population of HCPs, as stated in USP 39 [2].

Thus, comparing the coverage of various ELISAs – kits or custom – allows you to pick the best and most fitting ELISA for your drug.

Traditionally, you had to choose between two methods for HCP coverage evaluation. These were 2D gels followed by Western Blotting or immunoaffinity purification followed by 2D gel analysis.

However, both methods have widely known limitations, including manual and variable spot counting in gels [2, 3]. This led to improved coverage evaluation methods, such as ELISA-MS, which provides the name and identity of each HCP covered by the ELISA kit [4].

Coverage analysis with only 0.5 mg antibody – without a mock cell line

ELISA-MS combines ELISA-based immunocapture in the 96-well plate with LC-MS. The result is a list of individual HCPs in a drug sample recognized by the specific ELISA antibodies [4].

Where 2D gel-based and immunoaffinity chromatography-based coverage methods evaluate the antibodies outside of the ELISA plate, ELISA-MS mimics the conditions of the HCP-ELISA. Thus ELISA-MS captures the HCPs by the anti-HCP antibodies under the same conditions as the ELISA in the 96-well plate.

2D gel-based methods rely on spots in a gel to provide a total number of covered HCPs. If you only utilize this method, you cannot know which HCP the spot represents. And perhaps worse, you do not see the coverage of each HCP.

Moreover, column-based methods often need 10-15 mg ELISA antibodies. Whereas ELISA-MS only requires 0.5 mg antibody for running HCP coverage analysis and thus saves your valuable ELISA antibodies.

ELISA_MS principle
Illustration of the ELISA-MS™ method

With ELISA-MS, you can perform coverage analysis on an early process sample containing the drug-protein. Thus, you do not have to generate a mock cell line with the risk of having another HCP profile than your drug samples. This way, you can perform coverage analysis without investing time and money in developing the mock cell line.

Finally, when you get the lists of the individual HCPs covered in your process and the HCPs in the purified product, you will know if the HCP-ELISA detects the individual impurities in the product. Therefore, many consider ELISA-MS the best method for determining the coverage of ELISA kits.

You are free to use the best method – both for coverage and HCP analysis

We often get the question: Which coverage method do the regulatory authorities require?

The answer is that you have freedom of choice for the HCP analysis methods as long as you can demonstrate that the method is scientifically sound.

Are you interested in learning more about different HCP coverage methods to select the best ELISA kit? Then check out this webinar with HCP expert Rikke Raaen Lund:

References

[1]          Bracewell et al.: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control.” Biotechnology and Bioengineering2015

[2] U. S. Pharmacopeia: “USP 39 Published General Chapter <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals”. 2016

[3]          Zhu-Shimoni et al.: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods. “Biotechnology and Bioengineering2014

[4]          Pilley et al.: “A novel approach to evaluate ELISA antibody coverage of host cell proteins-combining ELISA-based immunocapture and mass spectrometry.” Biotechnology Progress. 2020

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Critical reasons why mAbs call for orthogonal HCP analysis techniques https://www.alphalyse.com/proteinanalysisblog/2021/05/21/orthogonal-hcp-method/ https://www.alphalyse.com/proteinanalysisblog/2021/05/21/orthogonal-hcp-method/#respond Fri, 21 May 2021 11:14:10 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2812 Question: We produce a monoclonal antibody (mAb) product, and our HCP ELISA shows low ppm levels. Recently, I heard that regulatory authorities might ask for orthogonal data on process-related impurities. Is this true? Why should we spend time and money on additional host cell protein analysis when our data shows our product is pure? Answer: […]

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Question:

We produce a monoclonal antibody (mAb) product, and our HCP ELISA shows low ppm levels. Recently, I heard that regulatory authorities might ask for orthogonal data on process-related impurities.

Is this true? Why should we spend time and money on additional host cell protein analysis when our data shows our product is pure?

Answer:

It is correct that mAb products are often relatively pure. This purity is due to the efficient removal of host cell proteins (HCPs) using protein A purification and polishing steps [1].

However, you should be aware that determining the HCP amount to a sparse sum does not rule out that the product may contain ‘problematic’ HCPs at low but critical levels.

Antibody

‘Problematic’ HCPs are those HCPs that may…

  1. Affect drug efficacy by reducing protein stability and potency,
    or
  2. Induce immunotoxic effects and immunogenic reactions in patients

-even in sub-ppm amounts.

Most people use HCP ELISAs to demonstrate process consistency and final-drug substance purity. However, fewer know that it merely monitors a subset of the HCP profile. Even with high coverage of the impurities in your sample, it may miss proteins that co-purify with the product or are poor immunogens due to low molecular weight [2].

Also, ELISA does not provide any details about the specific HCPs. Therefore, it is impossible to predict if any HCP (although at a low level) will cause problems later.

There are several examples where specific HCPs have delayed projects due to immunogenic reactions caused by particular HCPs. In other cases, products experienced reduced stability because of HCPs with enzymatic activity [3].

Hence, you need an orthogonal method to determine which HCPs appear in your product. Such a method could, for example, be Mass Spectrometry (MS).

ELISA kit for plant based expression system

Using mass spectrometry for HCP characterization

So how do you analyze your mAb for problematic HCPs?

Mass spectrometry as an orthogonal method for HCP analysis is very powerful. Mainly because it provides detailed information about the specific HCPs – information you cannot get from ELISA.

I.e., you can analyze the impurities to a level where you get the physical properties like molecular weight (Mw) and isoelectric point (pI). The analysis even provides the accession number to find information about the protein and related proteins. With this information, you are well equipped to evaluate if any of the HCPs are problematic.

The detailed information about each HCP can then be used for data-driven process optimization – to delete any problematic HCP.

Hence, it is crucial to use an orthogonal method to investigate if it contains any problematic HCPs. Even when your HCP ELISA shows low-ppm levels in your monoclonal antibody.

I recommend reading this interview with Denise Krawitz, PhD and principal consultant with CMC Paradigms LLC. It goes further into the managing of risks in HCP assay development.

Evaluate problematic HCPs in mAbs

Orthogonal method for evaluation of problematic HCPs in mAbs

Alphalyse has developed an orthogonal Mass Spectrometry method that lists the individual HCPs with the respective amounts – in any given sample [4].

Moreover, combining this standard method with a native digest optimized for mAbs, substantially lowers the detection limit, even down to sub-ppm levels.

As a unique service, you get a check for specific HCPs known to be of concern in mAb products. The assigned specialist also compares the results to the HCPs found in a study of commercially available mAbs.

You can use this information to remove specific HCPs and thus ensure the stability and efficacy of the drug. Similarly, you can lower the risk of immunogenic reactions in patients while providing regulatory authorities with extensive HCP data. 

Learn more on the Alphalyse website >

References:

[1]           Zhang et al.: ”Characterization of the co‐elution of host cell proteins with monoclonal antibodies during protein A purification,” Biotechnology Progress, 2016

[2]           Wang et al.: “Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk Assessment,” Biotechnology Bioengineering, 2009

[3]           Vanderlaan et al.: “Hamster Phospoholipase B-Like 2 (PLBL2): A Host Cell Protein Impurity in Therapeutic Monoclonal Antibodies Derived from Chinese Hamster Ovary Cells.” Bioprocess International, 2015

[4]           Heissel et al.: “Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein,” Protein Expression and Purification, 2018

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Investigation of ubiquitin in purified antibody products https://www.alphalyse.com/proteinanalysisblog/2021/03/30/ubiquitin-purified-antibody-products/ https://www.alphalyse.com/proteinanalysisblog/2021/03/30/ubiquitin-purified-antibody-products/#respond Tue, 30 Mar 2021 10:58:00 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2800 Question: Should we worry about ubiquitin in our mAb? Our process-specific HCP ELISA did not detect Host Cell Proteins (HCPs) in our purified monoclonal antibody (mAb). However, mass spectrometry-based HCP analysis identified ubiquitin in low-ppm amounts. Do you have any suggestions for why the Protein A purification did not remove it? Why did ELISA not […]

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Question:

Should we worry about ubiquitin in our mAb?

Our process-specific HCP ELISA did not detect Host Cell Proteins (HCPs) in our purified monoclonal antibody (mAb). However, mass spectrometry-based HCP analysis identified ubiquitin in low-ppm amounts.

Do you have any suggestions for why the Protein A purification did not remove it? Why did ELISA not detect it? And should we worry about its presence?

ubiquitin in antibody

Answer:

We saw the same problem in a recent client project and also observed ubiquitin in analyses of other mAb products. Therefore, it seems that it has several characteristics that makes it difficult to remove by regular mAb purification steps.

Before discussing these aspects, I would like to explain the characteristics and implications of ubiquitin. I.e., if you should worry about it altering your product or even harming the patient.

Ubiquitin – problematic or harmless?

Ubiquitin is a small regulatory protein of 76 amino acids. It alters protein fate by changing stability, function, subcellular localization, and interactions with other proteins [1-5]. When it is covalently attached to protein substrates, a process known as ubiquitination, it targets degradation by the ubiquitin-proteasome system. This post-translational modification is essential for cellular homeostasis.

With these abilities, it may seem unfavorable to have ubiquitin in the drug product. Nonetheless, no one has yet documented that it can cause immunogenic reactions in patients or affect drug stability.

Detection of ubiquitin in purified mAb product

You can investigate if ubiquitin is present in your samples and monitor its levels between batches with mass spectrometry data. Then – if you find it necessary – you can remove it based on its physicochemical properties.

Why was ubiquitin not removed during purification?

It is difficult to say why the purification process did not remove the ubiquitin. Perhaps it was covalently bound to the drug substance as a low-level post-translational modification. Or maybe it was just tightly associating with the mAb? It may happen since some HCPs attach non-covalently to the drug molecule as so-called hitchhiker HCPs. Alternatively, it may co-purify with the mAb if their physiochemical properties are similar. However, ubiquitin is a small protein of 8 kDa, whereas the mAb is 150 kDa. So this is not likely the problem.

Across the many mAbs analyzed by Alphalyse, we often identify proteins from the ubiquitin-related pathway. And the data indicate that ubiquitin binds tightly, covalently, or non-covalently to the mAb. This experience further supports that the otherwise efficient Protein A step does not remove ubiquitin as efficiently as other HCPs.

Ubiquitin detected in purified mAb

Why did the process-specific ELISA not detect ubiquitin?

In our client’s purified mAb, mass spectrometry (MS) determined the samples’ ubiquitin to be 1-25 ppm. The ubiquitin residual was thus within the detectable range of the ELISA. However, its relatively small size may be why ELISA missed it. ELISAs often have trouble identifying low Mw HCPs, since small Mw proteins are poor immunogens. I.e., they are not raising any high-specificity antibodies for the HCP ELISA [6].

A simple BLAST in UniProtKB reveals more than 250 variants from multiple organisms with above 90% sequence similarity. Furthermore, high homology could also play a role since ubiquitin conserves highly across species. This fact could make the CHO ubiquitin non-immunogenic in the rabbit used for raising the ELISA antibodies.

An analysis that detects low-ppm HCPs

To investigate the problem further, you may turn to mass spectrometry analysis.

Specifically, for mAb products, Alphalyse utilizes a highly sensitive method, which uses native digest to lower the detection limit.

You can learn more about the HCP analysis for monoclonal antibodies on the Alphalyse website >>

Protein analysis blog

References

  1.  Goldstein et al.: “Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells,” Proceedings of the National Academy of Sciences of the United States of America, 1975
  2. Wilkinson, KD: “The discovery of ubiquitin-dependent proteolysis,” Proceedings of the National Academy of Sciences of the United States of America, 2005
  3. Kimura et al.: “Regulatory mechanisms involved in the control of ubiquitin homeostasis,” Journal of Biochemistry, 2010
  4. Glickman et al.: “The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction,” Physiological Reviews, 2002
  5. Lin et al.: “Exploitation of the host cell ubiquitin machinery by microbial effector proteins,” Journal of Cell Science, 2017
  6. Bracewell et al.: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control,” Biotechnology and Bioengineering2015
ubiquitin in purified antibody

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Why measure process-related impurities in gene therapies with LC-MS? https://www.alphalyse.com/proteinanalysisblog/2020/12/02/cell-and-gene-therapies/ https://www.alphalyse.com/proteinanalysisblog/2020/12/02/cell-and-gene-therapies/#respond Wed, 02 Dec 2020 09:10:15 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2635 Question: Can I use mass spectrometry to document process-related impurities? I work in a biopharmaceutical company developing a gene therapy product based on an adenovirus vector. Recently, I have become responsible for setting up analytical methods, including impurity assays. However, it is difficult to find suitable methods since there are no good commercial HCP ELISA […]

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Question:

I work in a biopharmaceutical company developing a gene therapy product based on an adenovirus vector. Recently, I have become responsible for setting up analytical methods, including impurity assays. However, it is difficult to find suitable methods since there are no good commercial HCP ELISA kits for our human cell line.

So, can I use a method like mass spectrometry to document the process-related impurities?

Answer:

To put it simply: Yes, absolutely.

But let me explain further why mass spectrometry is a competent analysis method for protein impurities in cell and gene therapy products.

Research in the regenerative therapy field has advanced dramatically in the last few years. In 2018, FDA received 206 Investigational New Drug (IND) submissions, and FDA is expected to approve 10-20 cell and gene therapy products (GTPs) per year by 2025 [1, 2]. So, in response to these many new gene therapies and medicinal products (GTMPs) in development, FDA and EMA recently published new guidelines which advise developers to monitor process-related impurities [3, 4].

The manufacturing processes of GTPs are usually quite complex, and only small batches are produced for small patient populations [5]. The process uses new cell lines and ads various proteins from multiple sources and organisms during cell growth and harvest. These include bovine serum, human albumin, cytokines, antibodies, benzonase, etc.

Thus, a standard commercial ELISA cannot measure all these impurities. Also, it would be costly and challenging to develop a product-specific ELISA to cover the HCPs in these products. Luckily, mass spectrometry methods provide a more suitable analysis for GTPs.

(You can read more about the pitfalls of HCP ELISAs in this blog post.)

Now, let us have a closer look at the nature of cell and gene therapies

Cell therapy (also known as cellular therapy or cell transplantation) products contain a form of viable cells, e.g., CAR T-cells. These can come from a donor person (allogeneic cell therapy) or the patient himself (autologous cell therapy). They may even come from another species (xenogeneic cell therapy).

The cell types are, for instance, stem, progenitor, or primary cells. Often, the therapies are designed for neurological, autoimmune, cardiovascular, or ophthalmologic disorders [6, 7].

Gene therapy medicinal products (GTMPs or GTPs) consist of a carrier vector or delivery system. They typically contain a nucleic acid sequence, a virus, or a cell and classify as genetically modified organisms (GMOs).

The target can be specific tissues or cells to regulate, repair, replace, add, or delete a genetic sequence and affect a particular type of protein or class [6].

The two major GTP classes are recombinant viruses (viral vectors) and nonviral methods (e.g., plasmid DNA or bacterial vectors). The viruses carry human DNA to replace disease-causing genes in the patient. Retrovirus, adenovirus, herpes simplex, vaccinia, and adeno-associated virus (AAV) are highly effective and thus widely applied. However, nonviral methods may carry a lower risk of patient immunogenicity [8].

LC-MS – An orthogonal method for analysis of residual protein impurities in gene therapies based on adenovirus

So, let us talk about how you analyze protein residuals in products based on adenovirus expressed in a human cell line, e.g., HEK293 or A549 cells, and grown on a cell substrate containing serum albumin.

Adenoviruses comprise a protein capsid with different proteins enclosing the DNA and core proteins (See the figure below). When the virus is grown and purified from the human cell, the virus drug substance samples will also contain small amounts of residual human proteins. Thus, the high number of different proteins in the drug requires characterization by a highly sensitive approach before clinical administration [9-11].

Adenovirus structure

The best analysis method for this mixture is LC-MS, as it can both quantify and identify the different protein impurities. The SWATH LC-MS (liquid chromatography – mass spectrometry) analysis is a highly reproducible residual protein and HCP analysis method.

The SWATH LC-MS analysis provides A) The total amount of residual host cell proteins in ng/ml, B) A list of identified residual proteins and their amounts, and C) A list of identified viral proteins and their amounts.

Since the analysis is based on peptide analysis by LC-MS/MS, it does not rely on animal immune responses and can identify and quantify individual proteins – it is ideally suited for documenting process-related impurities in the complex GTP biopharmaceuticals [12, 13].

FDA guidelines for documentation of impurities in cell and gene therapies

Hopefully, you now have a basic understanding of the impurity analysis of GTPs. If you would like to learn more about how to apply mass spectrometry for impurity analysis of cell and gene therapy products, I recommend you read one of these articles:

>> BioPharm International: A Novel Method for Host Cell Protein Analysis

>> Biopharma from Technology Networks: A Smarter Way To Remove Host Cell Protein Contamination From Gene Therapies

Also, I have recorded a short webinar on FDA guidelines for monitoring impurities in cell and gene therapies:

References:

[1]          Lukashev et al.: ”Viral Vectors for Gene Therapy: Current State and Clinical Perspectives.”, Biochemistry (Moscow), 2016

[2]          Hernandez Bort et al.: ”Challenges in the Downstream Process of Gene Therapy Products,” American Pharmaceutical Review, 2019

[3]          European Medicines Agency: “Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products,” 2018

[4]          U.S. Food & Drug Administration: “Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),” 2020

[5]          Mavilio, F.: “Gene therapies need new development models,” Nature, 2012

[6]          Buzhor et al.: Cell-based therapy approaches: the hope for incurable diseases,” Regenerative Medicine, 2014

[7]          Cell therapy, Wikipedia

[8]          Nayerossadat et al.: “Viral and nonviral delivery systems for gene delivery,” Advanced Biomedical Research, 2012

[9]          Krawitz et al.: “Characterization of Residual Host Cell Protein Impurities in Biotherapeutics,” Analytical Characterization of Biotherapeutics, 2017

[10]          Rux et al.: “Adenovirus structure.”, Human Gene Therapy, 2005

[11]        Jin et al.: “Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins.Human Gene Therapy Methods, 2017

[12]        Wohlrab et al.: “Tracking Host Cell Proteins During Biopharmaceutical Manufacturing: Advanced Methodologies to Ensure High Product Quality,” American Pharmaceutical Review, 2018

[13]        Goey et al.: “Host cell protein removal from biopharmaceutical preparations: Towards the implementation of quality by design.”, Biotechnology Advances, 2018

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Speedy and detailed protein ID of SDS gel bands https://www.alphalyse.com/proteinanalysisblog/2020/06/09/protein-id-sds-gel-bands/ https://www.alphalyse.com/proteinanalysisblog/2020/06/09/protein-id-sds-gel-bands/#respond Tue, 09 Jun 2020 06:45:03 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2468 Question: Can I identify hundreds of gel bands in a few days? The biopharmaceutical company I work for consistently uses SDS PAGE and Western Blotting to check protein expression. But this means we spend a great deal of money on antibodies, which I feel may be unnecessary. In addition, we would like to identify several […]

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Question:

Can I identify hundreds of gel bands in a few days?

The biopharmaceutical company I work for consistently uses SDS PAGE and Western Blotting to check protein expression. But this means we spend a great deal of money on antibodies, which I feel may be unnecessary. In addition, we would like to identify several unknown proteins in the gels, possibly degradation products.

Is there a fast and reproducible method to identify hundreds of gel bands within a few days?

Answer:

If you develop or manufacture any protein-based biologic, you know it is essential to evaluate protein expression. Both to verify that the biopharmaceutical protein was produced in a sufficient amount and to investigate impurities co-produced by the host cell.

Fast protein ID of gel bands

The typical approach is to rely on one of these standard methods:

  • SDS-PAGE and Western Blotting (WB)

or

  • SDS-PAGE and In-gel digestion/mass spectrometry

The WB method is fast and easy to run if you have the antibody needed to recognize the drug product. However, since this approach depends on antibodies and their specificity toward the target protein, there is a degree of uncertainty [1].

Also, you should be aware that you rely on a constant antibody supply, making it costly when you have many samples. And it is vulnerable since you cannot analyze if you run out of antibodies. Furthermore, the antibodies (probably) will not cover the degradation products or other unknown proteins, making the approach incapable of identifying proteins in the sample beside the drug product [1, 2].

protein id from gel

Therefore, it makes sense to apply a method that runs without antibodies – and can take a high number of samples within a short time (hours and days). Some companies thus benefit from protein ID /confirmation by in-gel digestion and mass spectrometry (LC-MS). However, this requires more time than SDS-PAGE and WB – at least for traditional ways of setting up the method [1-3].

Improved speed and lower cost of running protein ID of gel-bands

Luckily, there is a way to reduce the time it takes to identify proteins in large numbers of gel bands using NanoLC-MS/MS: Adding an EVOSEP® one instrument in front of the mass spec instrument reduces the run time to 25% of traditional protein ID LC-MS. The gradient is 4x shorter without compromising protein detection and identification.

The setup combines robotic automation of the in-gel protein digestion and sample preparation for LC-MS. This results in higher speed, which is one of the most significant advantages compared to standard methods.

Furthermore, the approach overcomes problems with carryover, which is sometimes an issue with traditional LC-MS analysis [1-3]. We can thus increase reproducibility and sample throughput.

Identify impurities and degradation products

You will also obtain an overview of all proteins in your samples – not only confirming the drug product presence. You can even identify process-related impurities, such as residual protein added as part of the process development or host cell protein originating from the expression system (CHO, yeast, E. coli, etc.) used to produce the active pharmaceutical ingredient (API).

Finally, 20-80 percent sequence coverage is easily obtained and may be enough to distinguish isoforms and protein/peptide variants from each other. And the approach even identifies degradation products. This is because it does not rely on antibodies but determines all proteins in the sample independently of the process or the expression system.

Increase protein concentration on PVDF membrane

References:

[1]          Luque-Garcia et al.: “Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting,” Mol Cell Proteomics, 2008

[2]          Lahm HW, Langen H.: “Mass spectrometry: a tool for the identification of proteins separated by gels,” Electrophoresis, 2000

[3]          Ranjan AK, Gulati A: “Two-Dimensional Electrophoresis and Mass Spectrometry for Protein Identification,” Methods Mol Biol, 2019

Thomas Kofoed, Alphalyse
Thomas Kofoed, PhD

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Assessing and monitoring the quality of therapeutic mAb products https://www.alphalyse.com/proteinanalysisblog/2019/11/25/mab-characterization/ https://www.alphalyse.com/proteinanalysisblog/2019/11/25/mab-characterization/#comments Mon, 25 Nov 2019 13:09:54 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=2130 Question: Which analyses should be part of mAb characterization? I need to ensure the quality and consistency of my mAbs. Do you have any advice on which analyses I should conduct for proper mAb characterization? Answer: For several years, monoclonal antibodies (mAbs) have been the fastest-growing type of pharmaceutical molecules [1]: More than half of […]

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Question:

Which analyses should be part of mAb characterization?

I need to ensure the quality and consistency of my mAbs. Do you have any advice on which analyses I should conduct for proper mAb characterization?

Answer:

For several years, monoclonal antibodies (mAbs) have been the fastest-growing type of pharmaceutical molecules [1]:

More than half of all new drugs approved from 2015 to 2018 were mAbs [2].

Also, the number of approved mAb biosimilars has increased substantially during the last few years.

Before approval for clinical administration, you must show that you have characterized the biosimilar in detail, just like its originator product [2].

What I suggest

Characterization of mAbs can be pretty complex, though. It is not enough to check for the correct amino acid sequence – you must also analyze the structure of the N-glycans and the proper formation of disulfide bridges [2].

We suggest a comprehensive and robust mAb characterization program based on intact mass analysis and peptide mapping to get the complete picture.

A mAb characterization program often consists of the following steps:

Intact mass analysis

  • Can be used before and after reduction
  • Applicable before and after removal of N-glycans
  • As subunits after IdeS treatment

Peptide mapping

  • I am applying single or multiple digests using proteases, such as Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, etc.
  • Digestion under reduced and non-reduced conditions

Data analysis

  • Detailed characterization is based on the experimental data obtained by intact mass analysis and peptide mapping.
  • A high level of protein chemistry expertise is necessary for this process.

This workflow provides a reproducible, accurate, sensitive, and fast structural assessment.

The solution is particularly beneficial to the biopharmaceutical industry. Pharma companies thus use it to control mAb product quality and consistency, batch-to-batch comparability, and process modification.

For more information about characterization analysis, I suggest you visit our page dedicated to antibody analysis. Here you can learn about the different studies, e.g., assessing modifications like glycosylations and quantifying protein.

Amino Acid analysis

Related blog posts:

References:

[1]          Liu, J.K.H.: “The history of monoclonal antibody development – Progress, remaining challenges, and future innovations,” Annals of Medicine and Surgery, 2014

[2]          Walsh, G.: “Biopharmaceutical benchmarks 2018,” Nature Biotechnology, 2018

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How to document removal of a process related impurity https://www.alphalyse.com/proteinanalysisblog/2019/09/25/process-related-impurity/ https://www.alphalyse.com/proteinanalysisblog/2019/09/25/process-related-impurity/#respond Wed, 25 Sep 2019 07:24:26 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=1917 Question: How do I detect process-related impurity at low ppm levels? My company develops and produces a biologic. Recently, regulatory authorities asked us to document how efficiently downstream purification reduces specific enzymes added during process development. How can I measure and follow the levels of enzymes and other proteins added during manufacturing? Is a reproducible […]

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Question:

My company develops and produces a biologic. Recently, regulatory authorities asked us to document how efficiently downstream purification reduces specific enzymes added during process development.

How can I measure and follow the levels of enzymes and other proteins added during manufacturing? Is a reproducible method available that detects any process-related impurity down to low ppm levels?

Answer:

Yes, there is a terrific method for process-related impurity detection, with detection limits down to low ppm levels. It can track and quantify the residual protein through all purification steps and, eventually, the final biopharmaceutical product.

However, let’s first look at process-related impurities often applied in process development – and why they increasingly concern FDA/EMA.

Process-related impurities follow along with the product during the development and production of biologics, such as monoclonal antibodies, antibody-drug-conjugates, therapeutic proteins, gene therapy products, and vaccines.

Typically, we divide impurities into three subtypes that depend on origin:

  • Cell substrate-derived / product-derived (e.g., host cell proteins, host cell DNA)
  • Cell culture-derived (e.g., antibiotics, IPTG, DTT, growth factors)
  • Downstream derived / process-derived impurities (e.g., enzymes, buffer components)

Note that the third subtype often includes the cell-culture-derived impurities. We also call the third subtype process-related residuals.

Process and product related impurities in antibodies

This type consists of agents used to express and purify biological protein products. Standard process-related residuals include Benzonase nuclease, enzymes for site-specific PEGylation, aminopeptidase, Protein A, Tris, carriers, ligands, Tween/Polysorbate, DCA, TCEP, heavy metals, solvents, Triton-X, anti-foaming agents, PEI, TFA/Acetate, Imidazole, etc.

Why are residual impurities a concern?

Investigating the levels of residuals is crucial, especially in the final product sample. Because, like host cell proteins, residuals can influence the stability and efficacy of the active ingredient, and it may even pose a risk to the patient’s safety [1-3].

Since more sensitive analytical methods exist for residuals analysis, FDA and EMA seem more and more interested in data that map out the purification process’s efficiency [1-3].

FDA/EMA requests an increasing number of biopharmaceutical companies to document residual protein clearance before a biologic can move forward in late-stage clinical trials [1-3].

The problem, however, is that older methodologies like ELISA and HPLC are restricted by low antibody-specificity and too-high limit of quantification, respectively. Since the residuals in samples are present in only low-ppm levels, you need a highly sensitive and reproducible approach to get a detailed overview of the residual protein clearance [4, 5].

Luckily, SWATH mass spectrometry (LC-MS/MS) offers precisely that. It fragments all peptides instead of only a group of selected precursors using Data Independent Acquisition (DIA). SWATH LC-MS/MS divides the defined mass range into small mass windows for MS/MS fragmentation of the peptides. Afterward, comparing the acquired MS data to an ion library of known peptides will identify the proteins [6].

ELISA vs mass spectrometry

The advantages of SWATH LC-MS for residual protein analysis include:

  • Highly reproducible identification and quantification due to the use of the DIA mode
  • Interference from the high amount of drug substance on the signal of low abundant residuals is kept down by using small mass windows for MS/MS fragmentation.
  • Specific residuals can be followed throughout the entire process development. You can also use it to compare batches and for quality control after upscaling.
  • The high throughput of samples makes it possible to quickly assess many individual samples from various steps in the purification.
  • High sensitivity makes it possible to quantify low ppm levels.

With these advantages, SWATH LC-MS is ideal for analyzing process-related residuals in biologics development [6].

Check out a blog post about the SWATH methodology or download a poster for more details about Alphalyse’s process-related impurities analysis.

Using LC-MS to document process related impurities

References:

[1]          U.S. Department of Health and Human Services – Food and Drug Administration: Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection: Guidance for Industry,” 2019

[2]          U.S. Department of Health and Human Services – Food and Drug Administration: Q3C Impurities – Residual Solvents: Guidance for Industry“, 1997

[3]          European Medicines Agency: Guideline on Immunogenicity assessment of therapeutic proteins,” 2017

[4]          Zhu-Shimoni et al.: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods,” Biotechnology and Bioengineering2014

[5]          Bracewell et al.: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control,” Biotechnology and Bioengineering2015

[6]          Heissel et al.: “Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein,” Protein Expression and Purification2018

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Can I use commercial HCP ELISA kits in clinical trial documentation? https://www.alphalyse.com/proteinanalysisblog/2019/07/10/hcp-elisa-kits/ https://www.alphalyse.com/proteinanalysisblog/2019/07/10/hcp-elisa-kits/#respond Wed, 10 Jul 2019 08:13:32 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=1860 Question: Does our generic HCP ELISA kit have sufficient coverage? My company uses a commercial HCP ELISA kit to analyze clinical phase 1 material host cell protein. We hear that we must develop a process-specific ELISA for clinical phases 2 and 3. Developing a process-specific ELISA could take two years, cost a fortune, and might […]

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Question:

Does our generic HCP ELISA kit have sufficient coverage?

My company uses a commercial HCP ELISA kit to analyze clinical phase 1 material host cell protein. We hear that we must develop a process-specific ELISA for clinical phases 2 and 3. Developing a process-specific ELISA could take two years, cost a fortune, and might not even have good coverage.

Is there a way to ensure that we pick a commercial HCP ELISA with high coverage? And thus increase the chances of using it for approval of clinical-stage trials.

Answer:

The short answer is – yes.

Many people still believe clinical trials require the tiresome development of process-specific ELISA antibodies – and is unavoidable. It is probably because commercial HCP ELISA kits sometimes have a low HCP coverage*. (*the percentage of host cell proteins (HCPs) recognized by the antibodies [1]).

ELISA HCP coverage

A suitable assay could be any HCP ELISA documented to cover enough HCPs

However, recent recommendations from government officials at FDA and EMA suggest otherwise. They only say that the chosen HCP assay should have sufficient coverage with demonstrated ability to detect most HCPs present in the product [2].

Based on this, a suitable HCP coverage assay could be any ELISA documented to cover enough HCPs. And thus, even a commercial HCP ELISA with proper validation of its coverage [3], like CHO, E. coli, and HEK 293.

hcp-coverage

Now, let’s compare methods for evaluation of HCP ELISA kits:

Typically, you evaluate the coverage by protein spot counting, using 2D SDS-PAGE and Western Blotting, or Immunoaffinity binding and 1D/2D SDS-PAGE [3].

This method’s disadvantages are long: It includes high variability, low reproducibility, and a high risk of not detecting abundant HCPs of low MW. Furthermore, the HCP antigens denature during 2D PAGE analysis. In contrast, the method detects antigens in their native form in ELISA [4].

So what if a biopharmaceutical company wishes to skip process-specific antibody development? Then it needs a better coverage analysis to determine the precise coverage percent of the ELISA for specific HCPs. It enables comparing and selecting the ELISA with the best coverage of all HCPs,  whether commercial or process-specific [3, 4, 5].

The ideal coverage method includes a complete list of HCPs covered by the assay to select the best HCP ELISA [3, 4].

Fortunately, a method that can achieve this is now available and described below:

What is ELISA-based immunocapture?

This method first immobilizes the antibodies to the 96-well plate, like the sandwich ELISA. Subsequently, process samples are added, and antigens bind to their respective antibodies – if the HCP ELISA antibody kit contains them. Finally, the process digests the bound antigens with proteolytic enzymes before mass spectrometry (MS) sample prep and LC-MS/MS analysis.

Information-dependent acquisition (IDA) liquid chromatography (LC) tandem mass spectrometry (MS/MS) can find the proteins covered by the HCP ELISA. This method is denoted IDA LC-MS/MS or SWATH LC-MS/MS and uses the Sciex TripleTOF 6600 system. It also includes searches of relevant protein databases.

The analysis provides you with:

  • A list of proteins recognized by the ELISA antibodies
  • A list of all proteins in mock or process sample
  • The HCP coverage percentage
  • The specific coverage of HCPs in the drug product
Coverage analysis - a method for selecting the best ELISA kit

What else should you know about evaluating the coverage of HCP ELISA kits?

The new analysis was first introduced at BEBPA 2019 conference, the largest HCP conference in the world. This poster describes the method in more detail:

HCP COVERAGE POSTER

More information about the coverage analysis is available on the Alphalyse website.

References

[1]          Bracewell et al.: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control,” Biotechnology and Bioengineering2015

[2]          The United States Pharmacopeia: “Residual Host Cell Protein Measurement in Biopharmaceuticals,” USP 39 – NF 34, 2016 

[3]          Alphalyse: “BEBPA 2019 review – key topics and take-home messages“, 2019

[4]          Zhu-Shimoni et al.: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods,” Biotechnology and Bioengineering2014

[5]          Wang et al.: “Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk Assessment,” Biotechnology and Bioengineering, 2009

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What are Host Cell Proteins (HCPs)? https://www.alphalyse.com/proteinanalysisblog/2019/01/10/what-are-host-cell-proteins/ https://www.alphalyse.com/proteinanalysisblog/2019/01/10/what-are-host-cell-proteins/#respond Thu, 10 Jan 2019 10:25:43 +0000 https://www.alphalyse.com/proteinanalysisblog/?p=1882 Question: What are Host Cell Proteins, and how are they measured? I recently met the term Host Cell Proteins (HCPs) in relation to process development and production of biopharmaceuticals. How dangerous are HCPs to patients, and how can HCP levels be measured throughout the purification steps? Answer: In therapeutic protein production of, e.g., vaccines, monoclonal […]

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Question:

What are Host Cell Proteins, and how are they measured?

I recently met the term Host Cell Proteins (HCPs) in relation to process development and production of biopharmaceuticals. How dangerous are HCPs to patients, and how can HCP levels be measured throughout the purification steps?

Answer:

In therapeutic protein production of, e.g., vaccines, monoclonal antibodies (mAbs), and antibody-drug-conjugates (ADCs), a wide range of impurities resides along with the biopharmaceutical product itself. The contaminants include host cell proteins (HCPs), nucleic acids, lipids, and DNA [1-3]. Various purification steps are needed before the clinical administration of the product.

After purification, the final product may still contain a (reduced) number of HCPs. Notably, specific HCPs might affect, e.g., protein stability or immunogenicity. Thus, HCPs are classified as the most critical process-related impurities in biologics [4-6].

Unwanted immunogenicity and decreased protein stability

It is well known that a difference between the foreign molecule and the system where it is introduced (such as a human) is associated with an increased risk of recognition by the immune system.

Due to the genomic variance between commonly applied protein production hosts, such as E. coli, yeast, the mouse myeloma cell line (NS0), and Chinese Hamster Ovary cells (CHO), most HCPs have the potential to generate an immune response in humans. For example, a study showed that a reduction in HCPs correlated with a decline in the release of specific inflammatory cytokines [4].

There is also a theoretical possibility that some unwanted HCPs may function in humans if similar enough to human homologs. Thus, we may see side effects of introducing these foreign proteins in humans [4].

To summarize, HCPs might influence:

  • the product quality by proteolysis, particle formation, or enzymatic modification
  • the process by the failure of a specific purification step
  • as well as pose a threat to the patient by immunogenicity or decreasing activity of the biologic’s active ingredient.

How to evaluate the risk of Host Cell Proteins

To reduce the possible damage caused by HCPs or evaluate the risk, it is necessary to identify and quantify the HCPs during the manufacturing processes and in the final product. It has previously been shown that HCPs found in purified products result from interactions with the product itself. This unwanted co-purification is product- and process-condition specific and can thus be reduced with knowledge about the different HCPs [5].

The drawbacks of using ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) is often utilized for HCP analysis because of its quick testing time and straightforward interpretation [1, 7]. Unfortunately, HCP ELISAs have several drawbacks, including the initial time it takes to develop and harvest antibodies and their ability to bind to their antigen (antibody specificity) [7].

When using ELISA, it is worth considering that anti-HCP antibodies can only be produced if immunizations with the HCPs result in an immunogenic response in the research animals. If the animal’s immune system does not detect the individual HCP, it cannot produce antibodies against it [7].

The LC-MS solution

Recently, a more comprehensive Host Cell Protein analysis has become available, based on an innovative LC-MS approach. Where current ELISA-based methods only present approximate information about HCP amount, this HCP analysis is as extensive and detailed as none before [8].

Using SWATH mass spectrometry results in information about the specific HCPs and their quantity in the biopharmaceutical product. Furthermore, the approach can analyze the effect of various purification processes and optimize them to avoid extensive purification matrices.

In addition, a new, ground-breaking HCP coverage analysis on the market determines the individual HCPs (and coverage percentage) covered by the anti-HCP antibodies in the HCP ELISA kit. The method is based on a unique combination of immunocapture and extremely sensitive LC-MS/MS. You can apply it to evaluate different HCP ELISA kits to choose the most accurate kit.

Check out this poster from the 2019 edition of the world’s biggest HCP conference, BEBPA, where the new analysis was presented for the first time:

Download the HCP coverage poster

References:

[1]          Wohlrab et al.: Tracking Host Cell Proteins During Biopharmaceutical Manufacturing: Advanced Methodologies to Ensure High Product Quality.” American Pharmaceutical Review, 2018

[2]          Dimitrov, Dimiter: “Therapeutic proteins.”, Methods in Molecular Biology, 2012

[3]          Goey et al.: “Host cell protein removal from biopharmaceutical preparations: Towards the implementation of quality by design.”, Biotechnology Advances, 2018

[4]          Wang et al.: “Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk Assessment.”, Biotechnology and Bioengineering, 2009

[5]          Bracewell et a.l: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control.” Biotechnology and Bioengineering, 2015

[6]          Guiochon et al.: “Separation science is the key to succesful biopharmaceuticals.” Journal of Chromatography A, 2011

[7]          Zhu-Shimoni et al.: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods.” Biotechnology and Bioengineering, 2014

[8]          Heissel et al.: Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.” Protein Expression and Purification, 2018

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