20634e.htm … · scenarios (e.g. to deliver an antigen covering a broad spectrum of hcp species)....

Reference: PA/PH/Exp. HCP/T (15) 1 1

XXXX:20634 2

2.6.34. HOST-CELL PROTEIN ASSAYS 3

This general chapter provides guidance for the development and validation of host-cell protein (HCP) assaysused to test products obtained by recombinant DNA technology. It does not exclude the use of alternativeapproaches that are acceptable to the competent authority. 4

INTRODUCTION 5

Host-cell proteins (HCPs) are process-related impurities derived from the host organism used for the productionof a medicinal product by recombinant DNA technology. In order to mitigate their potential adverse effects (e.g.immunogenicity), HCP content is reduced to the lowest possible level. 6

HCP clearance during the purification process must be assessed and the HCP content determined using an HCPassay that has been evaluated and validated for a given product. 7

The HCP acceptance limit, typically expressed in nanograms of HCP per milligram of active substance (ppm),must be justified with regard to the HCP clearance capacity of the purification process and with regard to thepotential impact of residual HCP on patients, taking into account the worst-case amount of HCP that could beadministered with the product. 8

HCPs are generally measured using an immuno-based assay containing, as reagents, the HCP antigen (HCPreference standard) and the corresponding polyclonal antibodies (antisera). Antisera must cover a broadspectrum of HCPs representative of the product concerned. 9

Sandwich-type enzyme-linked immunosorbent assays (ELISA) are the most commonly employed assays toquantitatively assess the level of HCPs. The sensitivity is the result of the observed cumulative responses ofmany individual HCPs in comparison to the response of an HCP reference standard. The use of orthogonalanalytical methods (e.g. electrophoresis, HPLC, western blot, mass spectrometry) is recommended to supportthe development and selection of the assay, as well as the characterisation of process HCPs. 10

ASSAY SELECTION 11

Several types of assays are available, with selection taking into account several factors, including the stage ofdevelopment of the product, the nature of the host cell and the protein immunogenicity, the expression mode, themanufacturing process, and prior knowledge. When selecting and developing the assay, its life cycle (e.g.reagent supply, consistency, assay validation, process change) must also be considered. 12

TYPES OF ASSAY 13

Process-specific assays 14

Process-specific HCP assays (also called product-specific HCP assays) are developed and validated taking intoaccount the specificity of the production process, and using the host organism expressing the recombinantproduct. 15

The antigen is derived from a mock run of the active substance manufacturing process (or a processrepresentative of it) up to a step capable of generating a broad spectrum of HCPs in sufficient quantities. 16

The antisera raised must cover a broad range of HCPs, in order to detect as many different HCPs as possibleand to also accommodate process variations. 17

Platform assays 18

Platform assays are developed by individual manufacturers and customised for their expression host andprocesses. The same sets of reference standards and reagents may be used to monitor HCPs in severalproducts manufactured in the same expression host, provided that upstream processes (and downstream, ifrelevant) are sufficiently similar for these products. 19

Generic assays 20

Commercially available HCP test kits are commonly referred to as generic HCP assays. They are intended towork broadly across similar expression hosts. Detailed information on the preparation of the assay may not be

20634E.htm http://pharmeuropa.edqm.eu/TextsForComment/NetisUtils/srvrutil_getd...

1 z 8 2015-03-30 15:23

scenarios (e.g. to deliver an antigen covering a broad spectrum of HCP species). For example, the antigen-containing cell culture supernatant may be harvested beyond the minimum level of cell viability in order to includemore cytosolic proteins, which are released by additional cell lysis. 38

Downstream 39

The immunogens derived from the upstream process are usually only minimally processed (filtration,concentration), in order to obtain a broad spectrum of HCPs. Further purification is generally not recommended.40

However, in cases where the antigens are not representative or do not yield a suitably broad HCP coverage,mixing of mock materials from different processing steps can be considered. Enrichment may also be achievedby pooling materials from mock fermentation or purification runs using different operating conditions, or fromselective purification steps (e.g. to reduce large amounts of the few immunodominant HCPs). 41

Cross-contamination with the protein of interest 42

The antigen must be produced in a manner that avoids contamination with even minute traces of the product inorder to avoid cross-reactivity with the polyclonal antibodies. 43

To achieve this goal, dedicated or single-use equipment is used as much as possible. Where multi-purposeequipment is used, it must be cleaned appropriately. In addition, the risk of contamination when filling or handlingthe antigen in the laboratory environment must also be considered. 44

Characterisation and testing 45

Before using the antigen for immunisation, the protein content is assessed (total protein assay) and the absence


2 z 8 2015-03-30 15:23

Immunisation 64

One of the challenges of the immunisation step is to generate a polyclonal antibody that is highly specific andsensitive for each of the antigenic proteins in the complex mixture of HCPs used as an immunogen. An animal’simmune response must be stimulated against both the stronger and weaker antigens. 65

An animal host that yields sufficient amounts of immune-specific immunoglobulin G (IgG) is selected. Rabbits,goats or sheep are the most common animal species for this purpose. 66

Where both the polyclonal capture and detection antibodies are from the same source, it can be assumed thatthey recognise different epitopes on the same HCP. Alternatively, polyclonal anti-HCP antibodies from differentanimal species may be used. Using several animals for a given species may reduce individual variations inimmune competence and provide additional response diversity resulting in maximised antibody coverage againstthe HCP antigens. 67

An immune response to a limited number of HCP antigens may be obtained rapidly, particularly when adjuvantsare used to boost the immune response. However, in complex mixtures, differential enhancement of the immuneresponse towards weaker antigens or those at lower concentrations may be necessary. 68

It usually takes several immunisations to reach a maximum immunological response, and depending on thefrequency of immunisation, the process can take 3-6 months to complete. 69

The progress of the immune response against HCPs for a given immunisation scheme must be monitored bydetermining the antibody titre using, for example, an ELISA, and by comparing the results of 1D or 2Delectrophoresis (after protein staining) with a western blot, where the polyclonal anti-HCP antibodies are used asprimary antibody. In practice, some minor proteins that elicit a strong immune response may not be visible in the


3 z 8 2015-03-30 15:23

implementing a manufacturing process change that may impact the suitability of the HCP reagent, or when a newlot of antibody reagent is purified from the initial serum. 86

Accuracy 87

Accuracy is demonstrated by spike/recovery analysis of the HCP reference standard in a relevant backgroundmatrix (e.g. the active substance or a sample from a relevant purification step). 88

Specificity 89

Specificity is demonstrated by the absence of interference from the matrix background (including the activesubstance). For instance, data from the accuracy study can be used to assess specificity. 90

Precision 91

As for any other quantitative assay, repeatability, intermediate precision and reproducibility are appropriatelyaddressed. 92

Quantitation and detection limits 93

Sensitivity is usually in the ppm range and is normally described through the quantitation limit (QL). QL is typicallydetermined by HCP spike recovery studies in the active substance or an appropriate sample matrix, and iscalculated from the minimal spike providing a response with predefined accuracy and precision from replicateanalyses. 94

Detection limit (DL) is often not determined (optional validation parameter). 95

Linearity 96


4 z 8 2015-03-30 15:23

Depleted reagents 115114

Process change 117116 HCP reference standard

119118

Anti-HCP antibody 121120

The protein concentrationof the new referencestandard is determinedusing the same method asfor the current referencestandard to ensure that theprotein concentrations arecomparable. 122

Total protein concentrationof the new antibody isdetermined. The finalassay concentration mustbe titrated for the new lotin order to achieve asimilar standard curve asfor the current lot. 123

The effects of processchange on the HCPcomposition of relevantprocess steps areanalysed by suitablemethods (e.g.1D-/2D-PAGE, westernblot, HCP assay). 124

Reagentcharacterisation125

Using suitable methods(e.g. 1D-/2D-PAGE,2D-DIGE), the similarity inprotein compositionbetween the new and

For biotinylated detectionantibodies, thebiotin:proteinstoichiometry is controlledand ensured to be similar

If the process changedoes not lead to a relevantchange in HCPcomposition, the currentHCP reagents are also


5 z 8 2015-03-30 15:23

Standard curves obtainedwith the new versus thecurrent reference standardare assessed forsimilarity. 136

A bridging study isperformed with testing ofrelevant process samples(e.g. purification stepsfrom harvest to the finalactive substance). In aside-by-side experiment,new antibodies mustdetect HCP levels atdifferent process stepswith the same or bettersensitivity as currentantibodies. 137

Relevant process samples(e.g. purification stepsfrom harvest to the finalactive substance) from thenew and the previousprocess are testedside-by-side. 138

If reagent characterisationand ELISA testingdemonstrate suitability ofthe new HCP referencestandard, the currentreference standard can be

If reagent characterisationand ELISA testingdemonstrate that the newantibody is suitable, thecurrent antibody can bereplaced. No revalidation

If for the new process, theantibody shows similar orhigher immunoreactivitycompared to the previousprocess, and the HCPassay shows adequate


6 z 8 2015-03-30 15:23


7 z 8 2015-03-30 15:23


8 z 8 2015-03-30 15:23

20634e.htm … · scenarios (e.g. to deliver an antigen covering a broad spectrum of hcp species)....

Documents