face

Quantify HCPs of concern

Quantification of specific host cell proteins of concern

Afraid that specific HCPs in your drug product may halt regulatory approval?

HCPs may influence the overall safety profile of product by:

  • Immunogenicity. May induce an immune response against the product by adjuvant effects.
  • Homology to endogenous human proteins. Immunogenecity to induce cross reactivity against the human protein.
  • Enzymatic activity. May cleave and modify the drug substance or constituents in the drug product.
  • Biological activity.
  • Homology to the drug substance protein.

We determine the quantity of your HCPs of concern by LC MS/MS analysis. For this purpose we identify unique signature peptides for the HCP of concern. We then use synthetic isotope-labeled signature peptides for MRM quantitation. The labeled peptides are spiked into the drug sample in known amounts. Standard curves are generated for linearity and quantitative analysis. The quantitation assay can be developed for in-solution samples. It can also be used for HCPs detected by 1D and 2D Western blots.

The service includes:

  • Absolute quantification of HCP of concern.
  • Identification of 3 unique signature peptides for isotope labelling.
  • Standard curves and linearity assessment for dilution series of drug substance and isotope labeled peptide standards.
  • Quantification by peak areas in MRM LC-MS/MS.

If you do not know the identity of the host cell proteins in your samples, we can identify the HCPs prior to quantification. Take a look at our services for Identification of HCPs in pre-clinical and clinical batches.

How a customer used this service

One of Alphalyse’s biopharma customers produced a recombinant human protein in e. coli. In 2D Western Blot HCP antibodies recognized a HCP in exactly the same position as their drug substance protein. Using “Identification of host cell proteins >10 ppm” we identified an e.coli. HCP with very high sequence similarity to their drug substance protein. Hereafter, “Quantification of host cell proteins by AQUA peptide and MRM” showed that the sample contained 23 ppm of this specific HCP relative to their drug substance protein.

Services/Products
Protein analysis by Alphalyse

Quantification of host cell proteins of concern by MRM LC-MS/MS

  • Quantification of specific HCPs of concern
  • Linearity curves for isotope peptides and DS
  • Quantification by MRM LC-MS/MS
  • Requires pre-identification of HCP

$3,000.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote

Introduction

The absolute quantification of specific protein in complex matrices are based on the selection of specific peptides as signature peptides and used for assay development based on Multiple-Reaction-Monitoring (MRM). The project are typically divided into three parts.

Part 1: Feasibility study

The purpose of the feasibility study is to investigate the possibility to develop an assay that can be used for the quantification of the specific protein in the matrix.

The feasibility study will include:

  1. Planning phase – evaluate potential sample preparation protocols and signature peptides, i.e. specific marker peptides, based on the protein sequences.
  2. Digest and LC-MS/MS analysis to ensure that specific signature peptides are formed and that this/these peptides do not appear as part of miss-cleaved peptides or modified peptides (oxidized, deamidation etc.). The digest will be performed testing a number of different protocols including a range of proteases.
  3. MRM analysis of the selected peptides on Triple Quad instrument to verify formation of suitable MRM transitions.
  4. Short report evaluating the possibility for Alphalyse being able to develop an assay that can be used for the purpose of the project including an estimation of LOQ and LOD.

Part 2: Assay development

  1. Optimizing the sample preparation and digest of the proteins for mass spectrometric identification of released peptides.
  2. Ordering of isotope labeled peptide standards (AQUA peptides).
  3. Sample preparation and digest of the protein with addition of AQUA peptides for quantitative measurements. Selection of optimal MRM transitions for each peptide. Evaluate quantitative results and select quantitative peptides for quantification assay.
  4. Integrate workflow; sample prep. incl., reduction/alkylation, protein digestion, peptide cleanup, LC-MS/MS analysis and MRM measurement. Quantitative measurements using AQUA peptides and external calibration curves on the protein reference standard.
  5. Qualification of quantification assay; demonstration of measurement range, linearity, precision and accuracy.

Part 3: Sample analysis.

  1. Using the final assay to measure real samples.

Introduction
Protein quantification by mass spectrometry requires a protein in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

To develop MRM quantification assays requires the protein in pure form and a matrix sample without the protein similar to the matrix of the real samples to be analyzed.

Protein samples can be submitted in liquid or lyophilized.

  1. Purify the protein

    1. The chromatographic protein/peptide purity should be >90%
    2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents
    3. Minimum amount ~ 1 mg
  2. Put the protein sample into microcentrifuge tube

    1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
    2. Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
    3. Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample

Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork

Order analysis now
Newsletter Subscribe to the Alphalyse Newsletter to get 3 tips
about protein analysis methods and applications
Private, secure, spam-free
© Copyright Alphalyse , Inc. 2002 - 2017. All rights reserved.