Recombinant Factor C

Abstract

Recombinant proteins are the fundamental product of modern biotechnology. LAL is the purified protein extracted from horseshoe crabs used to test pharmaceutical products for bacterial endotoxins; the use of LAL has ensured the safe supply of medicine for over 30 years. A synthetic recombinant protein Factor C (rFC), the first protein in the LAL cascade, has been commercially available for 15 years, however market uptake has been limited for various reasons. With respect to horseshoe crab conservation and for other species that depend on the horseshoe crab, including the threatened Red Knot shorebird, the use of rFC for the detection of bacterial endotoxins was successfully validated for use with multiple pharmaceutical products. A validation strategy is described herein and an example validation protocol is provided for the convenience of the end-user.

Appendix I: Example Alternative Validation Protocol

The following protocol is an example product-specific validation worksheet using the alternative end-point fluorescence technique with rFC reagent. The protocol is provided to aid users in developing their own protocol, and is predicated on a documented holistic validation strategy.

1.1 General Information

Method code	[Local Method Code]
Test type	Safety
Analyte(s)	Bacterial endotoxins
Product matrix	[Insert Product Matrix]
Lab performing validation	[Insert Lab]
Principle of procedure	Fluorescence end-point using recombinant Factor C (rFC) reagent for the detection of bacterial endotoxins
Validation protocol	General Validation Protocol for [Insert Local Method] by Fluorometric Analysis ([Insert Local Validation Strategy Document])
Method	[Insert Local Method]

This document must be completed with the compound specific information, signed and dated by the author, technical reviewer, management approver, and quality approver prior to execution of the experiments.

1.2 Purpose

This document provides the compound specific information relating to the validation of the method for this product and the approval to use the general validation protocol listed above for this method validation. The combination of these two documents meets the requirements of [Insert Applicable Quality SOPs].

The general validation protocol ([Insert Local Validation Strategy Document]) describes the experiments and acceptance criteria to validate the fluorescence end-point method (rFC) for the detection of bacterial endotoxins. This document provides the additional information needed to validate for a specific item with experimentally pre-determined product specific preparation instructions, which includes an optimal sample test concentration (dilution factor) and test sensitivity in relation to the product limit, the maximum valid dilution (MVD), and other relevant sample preparation details (e.g., diluent, etc.)

1.3 Test Information

Sample(s) to be used in the experiments are listed below.

Lot numbers will be documented at the time of execution.

The validation samples are representative of the current manufacturing process.

MVD = (Limit × Sample Concentration)/Assay Sensitivity

Result Calculation = (Raw Result × Dilution Factor)/Sample Concentration

1.4 Standard Curve Preparation

Prepare the 0.01 λ standard curve as directed in [Insert Local Method] using the assay sensitivity as described in Test Information, or using the following example³:

• 1.0 EU/mL:100 μL of 20 EU/mL + 1900 μL of LRW

• 0.1 EU/mL:200 μL of 1.0 EU/mL + 1800 μL of LRW

• 0.01 EU/mL:200 μL of 0.1 EU/mL + 1800 μL of LRW

An example preparation of spiking solutions for use in the high and low spike accuracy and precision experiments is below:

• 3.16 EU/mL:316 μL of [20 EU/mL] + 1684 μL of LRW

• 0.316 EU/mL:200 μL of [3.16 EU/mL] + 1800 μL of LRW

1.5 Sample Preparation

For each of three independent assays, prepare a sample of each lot as follows. Fresh preparations are made for each independent assay.

Solution	Preparation	Dilution Factor	Conc. (mg/mL)
A
B
C
D

1.6 Positive Product Control (PPC)

Prepare the PPC as directed in [Insert Local Method] using Solution D above and the 1.0 EU/mL standard described in Test Information.

1.7 Accuracy and Precision

Prepare the accuracy and precision experiment samples by spiking 10 μL of the 3.16 EU/mL (high) and 0.316 EU/mL (low) spiking solutions into Solution D aliquots in the test plate.

1.8 pH Suitability

Determine item pH suitability by evaluating the item pH of the neat sample solution, diluted test solution, or the diluted test solution combined 1:1 with rFC reagent. Once the pH is shown to be within the acceptable range of 6.0–8.0, it is unnecessary to test subsequent solutions.

1.9 Analysis and Documentation of Results

Documentation of the execution of this protocol may be recorded in printed copies of the protocol attachment. Each independent sample analysis must meet all data acceptance criteria for the result(s) to be considered valid and acceptable for use in the validation documentation. Report results as per software report. [Insert Local Investigation Scenario Requirements].

1.10 Data Acceptance Criteria Per Assay

Parameter	Acceptance criteria	Rationale
r	0.980–1.000	Criteria from compendia and per Global Method
y-intercept	2.500–5.000	Criteria from reagent supplier and per Global Method
Slope	0.760–1.110	Criteria from reagent supplier and per Global Method
PPC	50–200%	Criteria from compendia and per Global Method
%CV of top standard	<25.00%	Criteria from reagent supplier and per Global Method
% CV of middle standard	<25.00%	Criteria from reagent supplier and per Global Method
% CV of low standard	<25.00%	Criteria from reagent supplier and per Global Method
%CV of sample	<25.00%, if applicable	Criteria from reagent supplier and per Global Method
%CV of PPC Sample	<25.00%	Criteria from reagent supplier and per Global Method
pH	pH of test article at time of assay must be within 6.0–8.0.	Criteria from reagent supplier and per Global Method

1.11 Validation Criteria

Parameter	Acceptance criteria	Rationale
Accuracy	Average recovery (%) of each challenge concentration must be NLT 50% or NMT 200% of theoretical concentration	[Insert Local Validation Strategy Document]
Precision	Precision of each challenge recovery (%CV) must be <25.00% Based on final average of each spiked concentration (high and low); calculated and reported in the validation report	[Insert Local Validation Strategy Document]
Inhibition/Enhancement	Positive product control (PPC) recovery is 50–200%	[Insert Local Validation Strategy Document]
pH	pH value must be 6.0–8.0	[Insert Local Validation Strategy Document]

1.12 Analysis of Failed Results

If the validation criteria are not met a technical review of the results will be initiated to determine appropriate action.

A qualified individual will evaluate the data to determine if the acceptance criteria outlined in this protocol have been met. If the validating lab does not meet the criteria set forth in this protocol, an investigation and any supplemental actions will be conducted as per local procedure.

1.13 Departures from Protocol

Summarize any protocol departures or investigations including whether data were accepted or not with rationale and references to the detailed information as per local standard operating procedures.

1.14 Reasons for Revision

Version	Reason	Author
0